Exp. Eye Res. (1979) 28, 483-500
Soluble Proteins of Intact Bovine Rod Cell Outer Segments WALTER
GODCHAUX
III
AXD
WILLIAM
F. ZIMMERMAN
Department of Biology, Webster Center for the Biologicd Sciences Amherst College, Amherst: J!lusx 01002. U.S.A. (Received 10 October 1978, Sew York) Centrifugation of a particulate fraction from homogenates of bovine retinas to isopycnic equilibrium on sucrose density gradients yielded two discrete bands of particles containing rhodopsin (rod outer segments). The band having the lower buoyant density appeared on microscopic examination to be isolated discs and fragments of outer segments and contained little protein other than rhodopsin. The band having a higher buoyant density appeared to be outer segments with intact plasma membranes and exhibited osmotic properties different from those of the disrupted outer segments. The intact outer segments were shown by gel electrophoresis to contain five major polypeptide species other than rhodopsin, amount,ing to about 3W’a of the total protein. These polypeptides did not separate from the rhodopsin of the intact outer segments during isopycnic or zone sedimentation in isotonic or hypertonic media, but could be extracted by hypotonic solutions, in which they remained soluble under conditions where all the rhodopsin-containing membranes were sedimented. The extracted proteins all sedimented at less than 30 S. and are regarded as soluble proteins contained within an osmotic compartment of intact outer segments. Three of the five polypeptides of these soluble proteins were found in only trave quantities among the soluble proteins prepared from the retina as a whole or from other particulate fractions; these proteins may be peculiar to the rod cell or its outer segment. The remaining two species were more widely distributed. Key u,ords: photoreceptors; rod outer segments; proteins (of out’er segments) ; rhodopsin; subcellular particles.
1. Introduction The rod cell outer segment consists largely of a vertical array of closely-packed discs. each a flattened vesicle surrounded by a unit membrane; the entire structure is surrounded by the plasma membrane (Young, 1969). It is generally accepted that, in rods, the visual pigment rhodopsin constitutes over 90:/A of the protein of the disc membranes (Heitzmann, 1972). L ess is known: however, about the presence and variety of nonmembrane proteins in the outer segment; these may he important in visual function (Frank and Buzney, 1975; Miki, Baraban, Kearns, Boyce and Bitenski. 1975). Preparations of rod cell outer segments, generally isolated by flotation in dense media after disruption of the retinal tissue, are important in the study of retinal function. Current uses of these preparations include investigation of the transduction between the photoisomerization of rhodopsin and the resulting nerve impulse (Hagins and Toshikama, 1974), the phenomena associated with the regeneration of rhodopsin (Rodieck, 1973; Zimmerman, Lion, Daemen and Bonting, 1975) and the turnover of proteins (Young, 1974; Papermaster, Converse and Siu, 1975). In all of these studies, the integrity of the isolated outer segments is potentially important. For example. studies of the enzyme activities present in the organelle (Frank and Buzney, 1975; Miki et al., 1975; Zimmerman et al., 1975) or of ion-transport phenomena (Bownds and Brodie, 1975; Hemminki, 1975) might yield results that depend heavily on the integrity of the plasma membrane and on the retention of any soluble contents that 0 1979 Academic Press Inc. (London) Limited
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might hc present in vivo. The lntrit,v of outer segment preparations is of I~CIIL;II importance, since activities present in the preparations might, in fact. l)e ;1wwi:rtc~cI with other particles present as cuntantinants. The int,egrity am1 Puritan OF is~~l;~tc~rl outer segments are usually characterized by electron microscopy ant1 IJV ashiivq Ii)r enzymes found in particles that’ arc potential contaminants (lYIc(~~~~~ncll~ 1!165: Papermuster mcl Dryer. 1974 ; Zimmerman, Daenran and Bont,ing? lYi6). I tulvpendent. and more general, criteria would frequently be useful. For these reasons, we undertook a study of the number and variety of proteins (other than rhodopsin) that are found specifically in intact rod outer segments. and have developed criteria for deciding whether or not individual polypeptides are native to that structure. Our approach was based on the observation t’hat intact and fragmented outer segments can be separated from ea,ch other, and from other part,iclcs. by centrifugation to buoyant equilibrium in continuous density gradient’s The polypeptides found in the fractions from these gradients were characterized u-it'll respect to amount and molecular weight 1)~ electrophoresis on po1yacrylan~itlr gels. A correlation between the amount of a particular polypeptidr and t,he a~tuoutn of rhodopsin in each fraction was taken as evidence for the association of the polypeptide with the outer segment. It was found that intact out)er segments (but not fragmented ones) contain substantial quantities of a few specific polypeptides other tha.n opsin; these were characterized as components of soluble proteins contained within an osmotic compartment. Other l~olyl~ept~ides were identified that are prol)al)ly contaminants. Workers using discontinuous gradients have previously observed apparent heterogeneity of the buoyant density of outer segments (McConnell, 1975; Papermaster and Dryer, 1974; Krebs and Kuhn, 1977) ; however, distributions in discontinuous gradients cannot, in themselves, provide evidence for the presence of two tliscrehe fractions of different buoyant density. Kuhn, Cook and Dreyer (1973) disrupted outer segments by sonication in a hypotonic buffer containing 10 nnvr-Tris-HC’l buffer this resulted in the solubilization of a and 5 m&r-ethylenediaminetetraacetate; number of polypeptides, some of which are probably the same as those wc have observed. However, additional proteins were present in their preparations. In the present report! it is shown that all the major nonopsin species can be solul~ilized simply by washing the outer segments with 5 mM-Tris-HCl buffer. We have also developed additional criteria that provide evidence for the normal localization of certain soluble proteins in the outer segment. 2. Materials and Methods Solutims
The Tris buffer was Tris-HCI, pH 7.2 at 20 rnM and 4°C. All sucrose and metrizamide concentrations are given as percent, w/v. Fractionatiola
of retinas
Bovine e?es were obtained from a local slaughterhouse, placed on ice in the dark within 20 mm of slaughter and dissected within 3 hr. All operations were performed at 4”C, and a dim red safelight was used during manipulation of preparations containing rhodopsin. Excised retinas were homogenized in 20“/o sucrose-20 miw-Tris buffer (2.5 ml, retina) using 6 strokes of a very loose-fitting Potter-Elvenhjem homogenizer operating at 300 rev/min. The homogenate was centrifuged for 4 min at 2OOxg in a horizontal
SOLUBLEPROTEIKS
OFROD OUTER SEGMEKTS
485
rotor, the pellet was resuspended by stirring in the 20% sucrose-Tris buffer (2.5 ml/retina) and the suspension centrifuged again under the same conditions. The supernatants were combined and centrifuged for 6 min at 7000 x g. The resulting pellet was suspended in the 20% sucrose-Tris buffer (2.5 ml/retina) by kituration with a wide-mouth pipet, again collected by centrifugation under the same conditions, and suspended in 65% sucrose,5 mlr-Tris buffer (0.8 ml/retina). The crude outer segment suspension Teas brought briefly to 25’C to reduce viscosity and forced gently 10 times through a 16-ga hypodermic needle. In some experiments, the supernatant from the initial ‘7OOOXg centrifugation was waa centrifuged at 12 OOUXg for 20 nun; the pellet, designated ~intermediate particles, washed once with the 20’$& sucrose--S mu-Tris buffer. The supernatant was underlayered rsith l-ml portions of 25 and 3076 sucrose+10 mu-Tris buffer and centrifuged at 150 000 ~g for 2 hr in an angle rotor. The supernatant was the soluble ,f~ncfio~a of the retiintl hornsgennte and the pellet, the nzicroso?& f~~fior~ Isopp
ic gradied
centrifugation
Suspensions of crude outer segments or of purified Srioose (hypertonic) gradients. fract.ions in 559: sucrose-5 m&f-Tris buffer were introduced at the bottom of 35-1111linear gradients from 27 to 50% sucrose containing 5 m&r-Tris buffer (5 ml suspension/gradient). The preparations were centrifuged for 16 hr at 80 000 Y g (4°C) in a horizontal rotor, and 1-ml fractions were collected. Sucrose-~nnetrz:zarrLide(isotokc) gradients. Purified outer segment fractions were suspended in 29” ’ 0 nletrizamidee2*3°/ ,,, sucrose-5 m&r-Tris buffer and introduced at the bot’tom of Il-ml linear gradients from 10% sucrose-5 rn>f Tris buffer to 15.5;; nletrizannde-6*4°; sucrose-5 m&r-Tris buffer (2 ml/gradient). The gradients were centrifuged for 2 hr at 160 ()Oc)xg (4°C) in a horizontal rotor, and 0.5 ml fractions were collected. ,411the solutions used were of physiological osmolarity, a condition made possible by the fact that metrizamide, which has the systematic name 2-(3-acetamido-5-N-methyl-acetami(lo-2,4,6,triiodobenzamido)-2-deoxy-o-glucose, has a higher molecular weight, and a lower partial sperifk volume, than sucrose (Rickmood and Rirnie, 1975). C’olkction.
and extraction
of particles
from
gw&wt
fractions
Sppropriate fractions from the isopycnic sucrose gradients were slowly diluted with cold 10’$0 sucrose-5 m&I Tris buffer to a final concentration of 20% sucrose and centrifuged for 20 min at lU OWJ Xg and 4°C. The pellets were drained thoroughly. For extraction of proteins, particles from the isopycnic gradient fractions were suspended in hypotonic buffer (5-m&f Tris buffer without sucrose; at least O-5 ml/mg protein) by trituration with a pipett#e, allowed to stand with occasional mixing for lo-15 min at W’C, and centrifuged for 20 min at 12 OOOXg. The pellet was re-extracted once, using the same procedure. The combined supernatants contained the extracted proteins. Intermediate particles and microsomes were extracted by the same procedure, except that in the latter case centrifuga,tion was at 150 OUOXg for 2 hr. Zone sedimentation,
Particles collected from isopycnic sucrose gradient fractions were suspended in lOql sucrose-5 mar-Tris buffer by trituration with a pipette. Portions (0.5 ml) were applied to 12 ml linear gradients from 15 to 2570 sucrose-5 mllr-Tris buffer with a cushion of 1 ml of c50”’!,I sucrose at the bottom. The gradients were centrifuged for 10 min at 2000Xg in a horizont.al rotor and l-ml fractions were collected. Extracts from particles (0.5 ml) were applied to 12 ml linear gradients from 10 to 25’& sucrose-5 rnbr-Tris buffer and centrifuged at 180 OOOxg in a horizontal rotor at 4°C. Frac:tions of 1 ml volume were collected.
G’el elect~o~kwesis Sodium dodecyl sulfate-polyacrylnmide gel electrophoresis was by the met ho(l 01 Laemmli, described in detail (as the alkaline discontinuous system) by JIaizel (1!471), and modified so that all solutions and gels contained 1 I-rwl-ethylellediulllilletetrilac. Separating gels (7 X 1lU mm) contained lop0 (\v./v) acrylamide and (j.35’: () (w/v) irlrtlr~lr~tlc~bisacrylamicle. Electrophoresis was carried out by 4 mA/pel until a phenol retl tr.aI,lcilq dye had migrated 100 mm. Gels were stained with Coomassie brilliaut blue, :mcl \\-(‘I’(’ calibrated with standard proteins consisting of bovine serum albumin (ant1 its rlinlcr), clehvclrogenase gamma globulin H nntl L chains, ovalbumin , glvceraldehytle-3-phosphate I . and myoglobin. DetewGaation
of protein and rhodopsin
Protein was determined using bovine serum albumin as standard. 111all Cases except the zone sedimentation of proteins, the method of Lowry, Rosebrough, Farr and Randall (1951) was used, modified so that reaction mixtures contained O*l(;;, (w/v) sodium dodecyl sulfate. For zone gradients of proteins, a dye-binding assay was used (Bradford, 1976). Rhodopsin was determined as absorbance at 500 nm after adjusting suspensions of particles to contain 0.70/, (w/v) hexadecyltrin~ethylann~~oniun~ &loride. Absorbance of representative samples was again determined after bleaching under imense light; residual absorbance, which was significant only in fractions of low rhodopsin content, was subtracted from the initial value. The extinction coefficient of rhodopsin was taken to be 40 000 1 11101-l clll-l. Histology Particles collected from isopycnic gradient fractions were fixed (as a pellet,) in 2Oq/, sucrose-20/b glutaraldehyde overnight at 4”C, postfixed in 207; sucrose-l% 0~0, for 1 hr at 4°C and dehydrated. The pellet was broken into small pieces, which were setlimented and embedded in Epon 812. Thin sections mere stained with lead citrate and uranyl acetate.
Materials for gel electrophoresis and for the dye-binding protein assay were obtained from Bio-Rad, Inc. Sucrose was the ultra-pure density gradient grade from Schwarz-Mann. Hexadecyltrimethylammonium chloride was from Eastman Organic Chemicals. Xetrizamide was from Nyegaard and Co., Oslo. Distilled and deionized water and reagentgrade chemicals were used in all cases.
3. Results Isolatiorb and morphology
of intact and disrupted
rod outer segments
Isopycnic gradient centrifugation of the particulate fraction of the retinal homoyielded two distinct rhodopsin-containing bands genate described under “Methods” (Fig. 1). Band 1 had a density of 1.127 g/ml and band II, a density of 1.142 g/ml. The occurrence in continuous gradients of two bands of particles containing rhodopsin has been noted previously (De Grip, Daemen and Bonting, 1972; Zimmerman et al., 1976; Krebs and Kuhn, 1977), but the present method of fractionation, with the sample applied in dense solution at the bottom of the gradient, provides better separation of the two bands from each other, and from denser particles, than we have bbtained before. On direct visual examination of the gradients, the two bands
SOLUBLE
PROTEINS
OF ROD
OUTER
SEGNENTS
487
appeared to be composed of finely-divided material. Examination under the phasecontrast microscope of wet preparations of samples taken from the gradients with a minimum of agitation revealed that all visible particles were dispersed; no aggregates were present. The wet preparations also showed that the particles in band II were larger than those in band I and were mostly rod-shaped, though they tended to become rounded -Density
tl
I.127 1 I
(g/ml)
I.140 I II
b---4
Fraction
I
number
FIG. 1. Isopynic sucrose gradient centrifugation of rod outer segment,s. Details are given under “Methods”. The densities shown were obtained by weighing measured volumes of the fractions indicated. The baby show fractions that were, in some cases, combined for further study. ( l ), Rhodopsin; (13) protein.
FIG. 2. Electron micrographs of the particles of band II(a) and band I(b). Particles were collected from fractions pooled as described under Fig. 1. Magnification is 9700x ; the bar indicates 1 pm.
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perhaps through swelling or folding- -upon standiug or upon riilutioil ant1 t#ion. Figure 2(a) shows an electron micrograph of a typical field of a s&ion f’ronl t.lrcs pellet of particles from band II. The particles were large (IL10 ~111in ;;cctiou). COIItwined the membrane lamellae t,ypicwl of outer segments and were sunour~rlctl ‘1~ an intact outer (plasma) membrane. Examination of numerous fields in several secttouh failed t’o reveal the presence of significant numbers of contanlinaCing pa’ticles exterior to the outer segment plasma membranes, nor did it show thr‘ prcencch of an! cilia. basal bodies. mitochondria or nuclei within bhe structures containing clip lamellac. The outer segment’s did contain some internal vesicles that, appearecl t,o I)(> more rounded than typical disc membranes: these were devoid of internal nleml)raneq and also of densely-staining contents and may have been swollen disc meml~~nt~s. In contrast to the particles of band II, t)hose of band 1 were smaller and appesretl to be mostly isolated groups of disc membranes not surrounded by an intact plasma membrane [Fig. f?(b)]. H ere, also: occasional small rounded vesicle,!: without obviou’: internal struct,ure, were present. C~‘lltl.ifll~ik
Protein
co&at
of the
roaouter
segment frnctiom
Band I (Fig. 1) contained 44 mg protein/pmol rhodopsin (range, 42 to 46 in replicate preparations). whereas band II contained 56 mg protein/pmol rhodopsin (range, 55 to 58). Gel electrophoresis of the tot’al prohein in particles collected from the pooled gradient fractions revealed that band I contained, almost exclusively, opsin (protein 37”) and its dimer (protein 74): trimer (protein 111) and, possibly, higher aggregates [Fig. 3(a), (b)]. Th e relative amount of multimeric forms was less when the amount of protein applied to the gels was decreased. Band I also contained traces of a few other species, notably protein 47. In contrast, band II contained more than trace amounts of a number of polypeptides other than opsin [Fig. 3(c), (d)]. The major species detectable were proteins 95, 53? 50 and 41. Particles were collected from the pooled gradient fractions, and protein was extracted from the particles with hypotonic buffer (5 mM-Tris buffer). Extraction of the particles of band II yielded 30% of the protein, and less than 2% of the rhodopsin, in the supernatant, whereas extraction of the particles of band I yielded less than 2% of the protein in the supernatant (Table I). Gel electrophoresis of these extracts [Fig. 3(g)] sh owed that proteins 95, 53, 50 and 51 were present in extracts from band II, but not band I, and these gels revealed the presence in band II of an additional polypeptide, protein 375, that was obscured by the dense opsin band in the gels of the total protein. The distinction between protein 378 and opsin is discussed further, below. Gel electrophoresis of the membrane sediment obtained after extraction of the particles of band II showed that most of proteins 95, 53, 50 and 41 had been removed [Fig. 3(f)], though it has been our experience that trace amounts of those species remain in the sediment even after extensive washing. Figure 4 shows a densitometric scan of the electrophoretic pattern of the proteins extracted from the particles found in band II. The amounts of individual species (as a percentage of the total soluble proteins assessed from the areas under the curve) were: protein 375, 28%; protein 41, 19%; protein 53 plus protein 50, 14% and protein 95, Sq&. Protein 375 does not separate from opsin on gel electrophoresis in detergent solution, but the two proteins are, nonetheless, distinct. Extraction of the intact * For brevity,
“protein
37” (for example) is used to refer to a polypeptide
of apparent mol. wt. 37 000.
SOLUBLE
PROTEINS
OF ROD
OUTER
489
SEGMEKTS
50
41 37
abcdef
g
h
i
j
k
I
%‘Iort,ions. Two minor components were also observed ; one that had a density Density 3 I.049 I
(g/ml) I.096 I
I.114 I
Fraction number
FIG. 10. Isopycnic centrifugation of intact and disrupted outer segments on isoto:lic gradients. Particles were collected from band II(a) and band I(b) of the preparative sucrose gradient and centrifuged on gradients of sucrose-metrizamide as described under “Materials and Methods”.
4Yti
W. GODCHAUS
III
AND
\C. F. ZIMIMERJIAX
slightly higher than the majcr component (this. also, contained the full cotnpletnr~nt of proteins) and one at a density of approximately 1.096 g/ml that was fonntl, cn gel electrophoresis, to be virtually devoid of proteins other than opsin. This tlrnsest COIW ponent probably resulted from disruption of some of the particles during manipulxtion. The particles of band I had a buoyant density of l-096 g/ml in thr isobmic gradient [Fig. IO(b)]. 4. Discussion The most likely explanation of the above observations is that rod outer segments contain, in addition to the membrane protein rhodopsin, substantial quantities of a few specific soluble proteins, amount’ing to about 309/o of the total protein of the organelle. The procedure used here for fractionation of retinas yields some rod outer segments that are morphologically intact and that contain the soluble proteins in an osmotic compartment, and some aggregates of disc membranes from outer segments that were disrupted during manipulation and have lost their soluble proteins. Occurresaceof soluble proteins in rod outer segments There is much evidence for the physical association of the soluble proteins 95, 53, 50, 41 and 3% with intact (band II) rod outer segments. These proteins were concentrated in fractions of the isopycnic gradient where the rhodopsin of intact outer segments was concentrated, and did not separate from the rhodopsin-containing structures during zone sedimentation of the particles. The buoyant density in sucrose gradients of disrupted outer segment material (band I) is, presumably, close to that of the disc membranes themselves. The greater density of the intact outer segments (band II) is consistent with the presence, in addition to the membranes. of an RSSOciatecl osmotic compartment cont’aining the soluble proteins (and possibly other macromolecules), highly concentrated hy virtue of the exposure of the particles to hypertonic sucrose solubions. Exposure of the intact outer segments to hypotonic media, a procedure that releases the soluble proteins from a particulate component, also converts the rhodopsin-containing structures to a form having the lower buoyant density. This provides an additional correlation between the hehaviour of the soluble proteins and that of the authent)ic outer segment structures. Also2 the soluble proteins banded with the rhodopsin in both the sucrose (hypertonic) and the isotonic gradients, in which the outer segments exhibited very different buoyant densiCes (and. therefore, particle volumes). It’ seems very unlikely that an unattached contaminating part’iole would have the same density as outer segments in the two different types of gradient. It seems probable that the soluble proteins were contained within the outer scgments and not within other subcellular structures that were adventitiously attached to their surface. Estimates of the concentration of rhodopsin in outer segmcrus range from 2 to 7 mu (reviewed by Rotlieck. 1973). The content of soluble prot’eins is about 17 mg/pmol rhodopsin (Table I). so their total concentration would tJe 34 to 120 mg/ml. Because of the presence of such large quantities of t,he soluble proteins, their presence in other particles would require that these particles be present in ntore than trace quantities. Furthermore. the soluble proteins present, in intact outer segments contained a limited collection of polypeptides; attached particles containing them would, presumably, have been limited to one or a very few types. Yet electron micrographs did not reveal the presence of large numbers of particles exterior tfo the outer segment plasma membranes. It is also unlikely that the proteins as such were
SOLUBLEPROTEINS
OFROD
OUTERSEGNENTS
497
adsorbed to the external surface of the plasma membrane of the outer segments, since simple transfer to a medium without sucrose (a change in osmotic pressure but not, in ionic strength) was sufficient to solubilize the proteins. It is also probable that proteins 95, 53, 50, 41 and 375 are native to the outer segment structures that were isolated, and were not introduced from the homogenate into the interior of the outer segments during breakage of the rod cell membrane. Three of the polypeptides extracted from the intact outer segments were found only a,s very minor components of the general soluble fraction of the homogenate or of ext.racts prepared from other particles, and t,hese preparat.ions contained species not present in the outer segments. This observation rules out the random inclusion of a portion of the external medium when the outer segment plasma membra,ne was llroken and resealed. Homogenization of retinas in ten times the usual volume of medium did not alter the amount or soluble protein content of band II outer srgrnents. It is therefore unlikely that the soluble proteins entered the outer segments 1)~.tliffl~sion front other phases of the homogenate. It has previously been suggested that the high buoyant density of’ some of t’hc outer segments might reflect the presence of a port’ion of the inner segment t,hat. remains attached to those structures (McClonnell, 1965). The cilium and its basal I)ody and the ellipsoid (mitochondrial) body are the inner segment structure lying nearest to the outer segment (Young, 1969),. we have been unable to observe these &u&ares in the sections of the particles of band II. It is possible, of course, that a portion of the cytoplasm of the inner segment not including these easily-identifiable st,ructures. was retained by the outer segments. However; the rarity of structures t,ypic.al of the inner segment in the sections a,ntl hhe finding of rather large quantities of soluble. prot,ein in the particles militates against the possibility that the prot,cins are derived exclusively from the inner segment. The question of whether any soluble priteins are confined exclusively to the outer segment cannot be addressed without means for examining specifically the contents of the inner segment; how-ever, three of the soluble proteins(95, 41 and 378) may well l)e peculiar either to the outer segment or to t)he rod cell. The general soluble fract’ion of the homogenate should be representative of the cytosol proteins of the several tlifferent, cell-types of the retina. Proteins 95, 41 and 37s were present, if a,t all, as \rery minor components of this fraction; such quantities as were present could have been derived from the disrupted outer segments or from the inner segment. Similarly, t,he three polypeptides are not major components of extracts prepared from other suhcellular fractions. There was some tendency for the three proteins to he present in hmall amounts in gradient fractions denser t)han band II (most noticeable for a l)rotein that may be 41, Fig. 6). This may have resulted from breakage of sonle outer segments-with deposition of their soluble probein--during passage through the gradient, or from the presence of small amounts of outer segments (difficult to detect a,t low concentration) in denser fractions. The former view is supported by the ohservation that the st)aining density of the protein hands decreased more with increasing density when extracts from particles collected by centrifugation (and thus freetl of extraneous soluble proteins) were examined (Fig. 7). Osmotic beheior
of the outer segments
The results obtained in this study strongly suggest that proteins 95, 53, 50: 41 ant1 378 are locat,ed in one or more solution phases confined within osmotic compartments
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that are present in the intact outer segrnent,s. disrupted by osmotic shock. at1~1alJs(bnt from the discs found in band I. There are other possible explanations, for cxutt~~~lc. that the proteins might be adsorbed to a membrane and released when the nlcn tl)rane is disrupted and the complex is exposed to media of low ionic st,rength. It is clificrtlt to understand how all five different polypept,ides could exhibit, the same wtlsorpticJt1 properties, however. Studies of the osmotic behavior of the outer segment fract’ions provide evidence for the existence of one or more osmotic compartments that are present in 6he int’act outer segments but not in the isolated discs. In the sucrose gradient, the intact, outer segments came to isopycnic equilibrium at a density of 1.142 g/ml, whereas their density in the isotonic gradient was 1.049 g/ml. If it is assunl& that the densit,) change resulted only from uptake of water, with no loss of solutes, then the volume of the particles in the solution of physiological osrnolarity was 190”,, greater than that in the 1.3 osmolar sucrose solution. The conclusion that such a change in volume took place is supported by the microscopic observations of t,he particles in solutions of the same osmolarities (Fig. 9) ; the average diameter of the particles in the solution of low osmolarity appears to be at least 1.5 times that in t*he solution of high osmolarity. In contrast, the discs (band I) had a density of 1.125 in the sucrose gradient (where they banded at an osmolarity of 1.14) and a tlensity of 1.096 in the gradient that was of physiological osmolarity. This corresponds to a volume increase of 30%; even if this is corrected to 35‘:,(,to account for the difference in the osmolarities at which the intact outer segments and the discs band in t(he sucrose gradient, it is small compared t#o the volume change of the intact. particles. The intact outer segments, therefore, contain one or more osmotic compartments that can be distinguished from the compartment within the discs themselves on the basis of behavior in solutiolis of clifferent osmolarity. The very small proporLiona1 increase in the volume of the discs between external osmolarities of l-14 and 0.32 would be explained if the disc vesicles contained a very small amount of solutes that contribute to the internal osmotic pressure; the discs lvould then be in virtually collapsed form at both these external osmolarities, as they appear to he in vivo (Young, 1969) where the external osmotic pressure should not exceed the physiological level. In fact, the discs can he observed to swell much more dramatically when transferred from 0.32 M-sucrose to the 5 mNl-Tris buffer without sucrose (assessed by measuring the volume of their pellet after centrifugation; unpublished results of the authors). This behavior is consistent with the ahove i&rpretation. Membranes obtained after extraction of the solul,le proteins from the intact outer segments with the 5 m;M-Tris buffer could still be observed to swell and shrink reversibly. Their internal compartment may therefore remain intact even during exposure to very low external osmolarities; presumably, the paucity of internal solutes results in an internal pressure insufficient to cause rupture of the membrane. For this reason, it seems unlikely that the proteins that were extracted by the dilute buffer were contained within the internal compartment of the discs themselves. Confinement of the soluble proteins within only the plasma membrane is sufficient to account for their behavior, but it is possible that yet a third osmotic compartment within the plasma membrane might contain the soluble proteins. Conceivably, the rounded vesicle observed in electron micrographs could play such a role, though these did not have densely-stained contents and also appeared to be present in the material from band I, that was essentially devoid of soluble proteins. If the compartment within the plasma membrane is, in fact, intact in the particles of band II, it must be
SOLUBLE PROTEIKS
OF ROD OUTER SEGMENTS
499
supposed either that the plasma membrane reseals after separation of the outer segment from the inner segment during disruption of the tissue, or else that there is a barrier between the two segments in vivo. Implications
for the physiological
study of outer segments and rhodopske
The integrity of isolated rod outer segments is of great importance to the study of their function in vitro. The methods described here provide a means of separating intact from disrupted outer segments and provide three criteria of integrity that are independent of microscopy : buoyant density, osmotic behavior and content of certain specific proteins. Purity of the preparations is equally important. The intact outer segment fraction, as prepared here, contains small amounts of proteins-most of them not extractable by hypotonic buffer-that show no correlation wyith rhodopsin in the density gradient fractions (e.g. protein 103). It must be suspected, at least, that these are contaminants. Assays for enzyme activities should be carried out on gradient fractions similar to those described here, to determine whether particular activities are in fact native to the outer segment. The re.sults of this study are consistent with the generally-accepted view that. rhodopsin constitutes over 90% of the membrane protein of outer segments (Heitzmann, 1972). However, gel electrophoresis in solution of strong detergents is currently t,he only practical means for assessing the freedom of rhodopsin preparations from small amounts of other proteins. Protein 37% and small amounts of protein 41 would he obscured by the opsin band in these gels (Fig. 4). These observations indicate the need for a cautious approach to the assumption that even well-washed membranes contain only a single protein, rhodopsin. Outer segment proteins are not synthesized locally, but. are transported from the inner segment (Young, 1974). We have recently investigated the incorporation of radioactive amino acids into protein in a cell-free system from bovine retina (Godchaux, 1978). The polypeptides synthesized by that system included (among others) species of molecular weight (15%) 50 000, 47 000, 41 000 and 36 000 (the system wa,s probably not active enough to complete many chains of mol. wt. 95 000). These four species accumulated at very high specific activity, and as completed chains no longer associated with transfer RNA, in particles that sedimented with the microsomal fraction. Three of the species (47, 41 and 36) were precipitated hy an antibody prepared against disc membrane protein that was undoubtedly contaminated with some of the soluble proteins described here [cf. Fig. 5(f)]. The labelled species of nominal mol. wt. weight 36 000 could have been opsin, protein 375, or a mixture of the two. This raises the interesting possibility, as a subject for future investigation, that some of the soluble proteins described in the present communication might be transported specifically to the outer segment and might be confined to a particulate compartment during that process.
ACKNOWLEDGMENTS The authors wish to thank Mr Glen Winkel for his excellent assistance in electron microscopy and Dr W. E. Robinson for suggesting the experiments with isotonic gradients. This work was supported by a grant (EY-00675) to W. F. Z. from the National Institutes of Health and by Institutional Funds (assigned to W. G.) granted to Amherst College by that agency.
REFERENCES Bownds, D. and Brodie, A. E. (1975). Light-sensit,ive swelling of isolated frog rod outer segme~~th as an in vitro assay for visual transduction and dark adaptation. J. (/e?b. Physiol. 66, 41)7 -S.i. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantit.iw of protein utilizing the principle of’ protein-dye binding. Analyt. Biochem. 72, 248~-54. De Grip, W. J., Daemen. F. J. M. and Bont)ing, S. L. (1972). Biorhemical aspects of the visu;~l process, XVIII. Enrichment of rhodopsin in rod outer segment membrane preparation. Vision Rev. 12, 1697-707. Frank. R. N. and Buzney, S. M. (1975). Mechanism and specificit,y of rhodopsin phosphorylat’ion. Biochem,istry 14, 5110-17. Godchaux, W. (1978). Biosynthesis of proteins in a cell-free syst,em from bovine retina. Bioche,,/. Biophys. Acta Ko. 99232, in the press. Hagins, W. A. and Yoshikami, S. (1974). A role for calcium ion in excitat’ion of retinal rods and cones. Exp. Eye Res. l&299-305. Hemminki, K. (1975). Accumulation of calcium by retina,1 outer segments. Bctrc Physiol. &and. 95, 117-25. Heitzmann, H. (1972). Rhodopsin is the predominant protein of rod outer segment membranes. Nature, New Biol. 235, 114. Jan, 1,. 1’. and Revel, J. (1974). Ultrastructural localization of rhodopsin in vertebrate retina. J. Cell Biol. 62, 257-72. Krebs, W. and Kuhn, H. (1977). Structure of isolated bovine rod outer segment membranox. Exp. Eye Res. 25,511-26. Kuhn, H., Cook, J. H. and Dreyer, W. J. (1973). Phosphorylation of rhodopsin in bovine photoreceptor membranes, a dark reaction after illumination. Biochemistry 12, 2495-502. Lowry, 0. H., Rosebrough, N. J., Parr, A. L. and Randall, R. J. (1951). Protein measurements with Polin phenol reagent. J. Biol. Chem. 193, 265-75. Maizel, J. V., Jr. (1971). Electrophoresis of viral proteins. Methods in Virology 5,180-247. McConnell, D. G. (1965). The isolat’ion of retinal outer segment fragments. J. Cell, Biol. 27,459-73. Miki, N.. Baraban, J. M., Kearns, J. J., Boyce, J. J. and Bitenski, M. W. (1975). Purification and properties of the light-activated phosphodiesterase of rod out,er segments. J. Biol. C~PYYI. 250,6320-27. Papermaster, D. S. and Dryer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 13, 243844. Papermaster, D. S., Converse, C. A. and Siu, J. (1975). Membrane biosynthesis in the frog retina: Opsin transport in the photoreceptor cell. Biochemistry 14, 1343-52. Rickwood, D. and Birnie, G. D. (1975). Metrizamide, a new density-gradient medium. Frd. F:ur. Biochem. Sot. Letters 50, 102-10. Rodieck, R. W. (1973). The Vertebrate Retina: Principles of Xtructure and Function. Pp. I 11-12. W. H. Freeman and Co., San Francisco. Saari, J. C. and Futterman, S. (1976). Retinol-binding protein in bovine retina: Isolation and partial characterization. Exp. Eye Res. 22, 425-35. Young, R. W. (1969). The organization of vertebrate photoreceptor cells. In The Retina: MorphoEogy, Function and Clinical Characteristics (Eds Straatsma, B. R., Hall, M. O., Allen, R. A. and Crescitelli, F.). Pp. 177-210. University of California Press, Berkeley. Young, R. W. (1974). Biogenesis and renewal of visual cell outer segment membranes. Exp. Eye Res. 18, 215-23. Zimmerman, W. F., Lion, F., Daemen, F. J. M. and Bonting, S. L. (1975). Distribution ofstereospecific retinol dehydrogenase activities in subcellular fractions of retina and pigment epithelium. Exp. Eye Res. 21, 325-32. Zimmerman, W. F., Daemen, F. J. M. and Bonting, S. L. (1976). Distribution of enzyme activities in subcellular fractions of bovine retina. J. Bill. Chem. 251, 4700-5.
Soluble Proteins of Intact Bovine Rod Cell Outer Segments WALTER
GODCHAUX
III
AXD
WILLIAM
F. ZIMMERMAN
Department of Biology, Webster Center for the Biologicd Sciences Amherst College, Amherst: J!lusx 01002. U.S.A. (Received 10 October 1978, Sew York) Centrifugation of a particulate fraction from homogenates of bovine retinas to isopycnic equilibrium on sucrose density gradients yielded two discrete bands of particles containing rhodopsin (rod outer segments). The band having the lower buoyant density appeared on microscopic examination to be isolated discs and fragments of outer segments and contained little protein other than rhodopsin. The band having a higher buoyant density appeared to be outer segments with intact plasma membranes and exhibited osmotic properties different from those of the disrupted outer segments. The intact outer segments were shown by gel electrophoresis to contain five major polypeptide species other than rhodopsin, amount,ing to about 3W’a of the total protein. These polypeptides did not separate from the rhodopsin of the intact outer segments during isopycnic or zone sedimentation in isotonic or hypertonic media, but could be extracted by hypotonic solutions, in which they remained soluble under conditions where all the rhodopsin-containing membranes were sedimented. The extracted proteins all sedimented at less than 30 S. and are regarded as soluble proteins contained within an osmotic compartment of intact outer segments. Three of the five polypeptides of these soluble proteins were found in only trave quantities among the soluble proteins prepared from the retina as a whole or from other particulate fractions; these proteins may be peculiar to the rod cell or its outer segment. The remaining two species were more widely distributed. Key u,ords: photoreceptors; rod outer segments; proteins (of out’er segments) ; rhodopsin; subcellular particles.
1. Introduction The rod cell outer segment consists largely of a vertical array of closely-packed discs. each a flattened vesicle surrounded by a unit membrane; the entire structure is surrounded by the plasma membrane (Young, 1969). It is generally accepted that, in rods, the visual pigment rhodopsin constitutes over 90:/A of the protein of the disc membranes (Heitzmann, 1972). L ess is known: however, about the presence and variety of nonmembrane proteins in the outer segment; these may he important in visual function (Frank and Buzney, 1975; Miki, Baraban, Kearns, Boyce and Bitenski. 1975). Preparations of rod cell outer segments, generally isolated by flotation in dense media after disruption of the retinal tissue, are important in the study of retinal function. Current uses of these preparations include investigation of the transduction between the photoisomerization of rhodopsin and the resulting nerve impulse (Hagins and Toshikama, 1974), the phenomena associated with the regeneration of rhodopsin (Rodieck, 1973; Zimmerman, Lion, Daemen and Bonting, 1975) and the turnover of proteins (Young, 1974; Papermaster, Converse and Siu, 1975). In all of these studies, the integrity of the isolated outer segments is potentially important. For example. studies of the enzyme activities present in the organelle (Frank and Buzney, 1975; Miki et al., 1975; Zimmerman et al., 1975) or of ion-transport phenomena (Bownds and Brodie, 1975; Hemminki, 1975) might yield results that depend heavily on the integrity of the plasma membrane and on the retention of any soluble contents that 0 1979 Academic Press Inc. (London) Limited
0014-4835/79/040483+18$01.00/O H*
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184
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might hc present in vivo. The lntrit,v of outer segment preparations is of I~CIIL;II importance, since activities present in the preparations might, in fact. l)e ;1wwi:rtc~cI with other particles present as cuntantinants. The int,egrity am1 Puritan OF is~~l;~tc~rl outer segments are usually characterized by electron microscopy ant1 IJV ashiivq Ii)r enzymes found in particles that’ arc potential contaminants (lYIc(~~~~~ncll~ 1!165: Papermuster mcl Dryer. 1974 ; Zimmerman, Daenran and Bont,ing? lYi6). I tulvpendent. and more general, criteria would frequently be useful. For these reasons, we undertook a study of the number and variety of proteins (other than rhodopsin) that are found specifically in intact rod outer segments. and have developed criteria for deciding whether or not individual polypeptides are native to that structure. Our approach was based on the observation t’hat intact and fragmented outer segments can be separated from ea,ch other, and from other part,iclcs. by centrifugation to buoyant equilibrium in continuous density gradient’s The polypeptides found in the fractions from these gradients were characterized u-it'll respect to amount and molecular weight 1)~ electrophoresis on po1yacrylan~itlr gels. A correlation between the amount of a particular polypeptidr and t,he a~tuoutn of rhodopsin in each fraction was taken as evidence for the association of the polypeptide with the outer segment. It was found that intact out)er segments (but not fragmented ones) contain substantial quantities of a few specific polypeptides other tha.n opsin; these were characterized as components of soluble proteins contained within an osmotic compartment. Other l~olyl~ept~ides were identified that are prol)al)ly contaminants. Workers using discontinuous gradients have previously observed apparent heterogeneity of the buoyant density of outer segments (McConnell, 1975; Papermaster and Dryer, 1974; Krebs and Kuhn, 1977) ; however, distributions in discontinuous gradients cannot, in themselves, provide evidence for the presence of two tliscrehe fractions of different buoyant density. Kuhn, Cook and Dreyer (1973) disrupted outer segments by sonication in a hypotonic buffer containing 10 nnvr-Tris-HC’l buffer this resulted in the solubilization of a and 5 m&r-ethylenediaminetetraacetate; number of polypeptides, some of which are probably the same as those wc have observed. However, additional proteins were present in their preparations. In the present report! it is shown that all the major nonopsin species can be solul~ilized simply by washing the outer segments with 5 mM-Tris-HCl buffer. We have also developed additional criteria that provide evidence for the normal localization of certain soluble proteins in the outer segment. 2. Materials and Methods Solutims
The Tris buffer was Tris-HCI, pH 7.2 at 20 rnM and 4°C. All sucrose and metrizamide concentrations are given as percent, w/v. Fractionatiola
of retinas
Bovine e?es were obtained from a local slaughterhouse, placed on ice in the dark within 20 mm of slaughter and dissected within 3 hr. All operations were performed at 4”C, and a dim red safelight was used during manipulation of preparations containing rhodopsin. Excised retinas were homogenized in 20“/o sucrose-20 miw-Tris buffer (2.5 ml, retina) using 6 strokes of a very loose-fitting Potter-Elvenhjem homogenizer operating at 300 rev/min. The homogenate was centrifuged for 4 min at 2OOxg in a horizontal
SOLUBLEPROTEIKS
OFROD OUTER SEGMEKTS
485
rotor, the pellet was resuspended by stirring in the 20% sucrose-Tris buffer (2.5 ml/retina) and the suspension centrifuged again under the same conditions. The supernatants were combined and centrifuged for 6 min at 7000 x g. The resulting pellet was suspended in the 20% sucrose-Tris buffer (2.5 ml/retina) by kituration with a wide-mouth pipet, again collected by centrifugation under the same conditions, and suspended in 65% sucrose,5 mlr-Tris buffer (0.8 ml/retina). The crude outer segment suspension Teas brought briefly to 25’C to reduce viscosity and forced gently 10 times through a 16-ga hypodermic needle. In some experiments, the supernatant from the initial ‘7OOOXg centrifugation was waa centrifuged at 12 OOUXg for 20 nun; the pellet, designated ~intermediate particles, washed once with the 20’$& sucrose--S mu-Tris buffer. The supernatant was underlayered rsith l-ml portions of 25 and 3076 sucrose+10 mu-Tris buffer and centrifuged at 150 000 ~g for 2 hr in an angle rotor. The supernatant was the soluble ,f~ncfio~a of the retiintl hornsgennte and the pellet, the nzicroso?& f~~fior~ Isopp
ic gradied
centrifugation
Suspensions of crude outer segments or of purified Srioose (hypertonic) gradients. fract.ions in 559: sucrose-5 m&f-Tris buffer were introduced at the bottom of 35-1111linear gradients from 27 to 50% sucrose containing 5 m&r-Tris buffer (5 ml suspension/gradient). The preparations were centrifuged for 16 hr at 80 000 Y g (4°C) in a horizontal rotor, and 1-ml fractions were collected. Sucrose-~nnetrz:zarrLide(isotokc) gradients. Purified outer segment fractions were suspended in 29” ’ 0 nletrizamidee2*3°/ ,,, sucrose-5 m&r-Tris buffer and introduced at the bot’tom of Il-ml linear gradients from 10% sucrose-5 rn>f Tris buffer to 15.5;; nletrizannde-6*4°; sucrose-5 m&r-Tris buffer (2 ml/gradient). The gradients were centrifuged for 2 hr at 160 ()Oc)xg (4°C) in a horizontal rotor, and 0.5 ml fractions were collected. ,411the solutions used were of physiological osmolarity, a condition made possible by the fact that metrizamide, which has the systematic name 2-(3-acetamido-5-N-methyl-acetami(lo-2,4,6,triiodobenzamido)-2-deoxy-o-glucose, has a higher molecular weight, and a lower partial sperifk volume, than sucrose (Rickmood and Rirnie, 1975). C’olkction.
and extraction
of particles
from
gw&wt
fractions
Sppropriate fractions from the isopycnic sucrose gradients were slowly diluted with cold 10’$0 sucrose-5 m&I Tris buffer to a final concentration of 20% sucrose and centrifuged for 20 min at lU OWJ Xg and 4°C. The pellets were drained thoroughly. For extraction of proteins, particles from the isopycnic gradient fractions were suspended in hypotonic buffer (5-m&f Tris buffer without sucrose; at least O-5 ml/mg protein) by trituration with a pipett#e, allowed to stand with occasional mixing for lo-15 min at W’C, and centrifuged for 20 min at 12 OOOXg. The pellet was re-extracted once, using the same procedure. The combined supernatants contained the extracted proteins. Intermediate particles and microsomes were extracted by the same procedure, except that in the latter case centrifuga,tion was at 150 OUOXg for 2 hr. Zone sedimentation,
Particles collected from isopycnic sucrose gradient fractions were suspended in lOql sucrose-5 mar-Tris buffer by trituration with a pipette. Portions (0.5 ml) were applied to 12 ml linear gradients from 15 to 2570 sucrose-5 mllr-Tris buffer with a cushion of 1 ml of c50”’!,I sucrose at the bottom. The gradients were centrifuged for 10 min at 2000Xg in a horizont.al rotor and l-ml fractions were collected. Extracts from particles (0.5 ml) were applied to 12 ml linear gradients from 10 to 25’& sucrose-5 rnbr-Tris buffer and centrifuged at 180 OOOxg in a horizontal rotor at 4°C. Frac:tions of 1 ml volume were collected.
G’el elect~o~kwesis Sodium dodecyl sulfate-polyacrylnmide gel electrophoresis was by the met ho(l 01 Laemmli, described in detail (as the alkaline discontinuous system) by JIaizel (1!471), and modified so that all solutions and gels contained 1 I-rwl-ethylellediulllilletetrilac. Separating gels (7 X 1lU mm) contained lop0 (\v./v) acrylamide and (j.35’: () (w/v) irlrtlr~lr~tlc~bisacrylamicle. Electrophoresis was carried out by 4 mA/pel until a phenol retl tr.aI,lcilq dye had migrated 100 mm. Gels were stained with Coomassie brilliaut blue, :mcl \\-(‘I’(’ calibrated with standard proteins consisting of bovine serum albumin (ant1 its rlinlcr), clehvclrogenase gamma globulin H nntl L chains, ovalbumin , glvceraldehytle-3-phosphate I . and myoglobin. DetewGaation
of protein and rhodopsin
Protein was determined using bovine serum albumin as standard. 111all Cases except the zone sedimentation of proteins, the method of Lowry, Rosebrough, Farr and Randall (1951) was used, modified so that reaction mixtures contained O*l(;;, (w/v) sodium dodecyl sulfate. For zone gradients of proteins, a dye-binding assay was used (Bradford, 1976). Rhodopsin was determined as absorbance at 500 nm after adjusting suspensions of particles to contain 0.70/, (w/v) hexadecyltrin~ethylann~~oniun~ &loride. Absorbance of representative samples was again determined after bleaching under imense light; residual absorbance, which was significant only in fractions of low rhodopsin content, was subtracted from the initial value. The extinction coefficient of rhodopsin was taken to be 40 000 1 11101-l clll-l. Histology Particles collected from isopycnic gradient fractions were fixed (as a pellet,) in 2Oq/, sucrose-20/b glutaraldehyde overnight at 4”C, postfixed in 207; sucrose-l% 0~0, for 1 hr at 4°C and dehydrated. The pellet was broken into small pieces, which were setlimented and embedded in Epon 812. Thin sections mere stained with lead citrate and uranyl acetate.
Materials for gel electrophoresis and for the dye-binding protein assay were obtained from Bio-Rad, Inc. Sucrose was the ultra-pure density gradient grade from Schwarz-Mann. Hexadecyltrimethylammonium chloride was from Eastman Organic Chemicals. Xetrizamide was from Nyegaard and Co., Oslo. Distilled and deionized water and reagentgrade chemicals were used in all cases.
3. Results Isolatiorb and morphology
of intact and disrupted
rod outer segments
Isopycnic gradient centrifugation of the particulate fraction of the retinal homoyielded two distinct rhodopsin-containing bands genate described under “Methods” (Fig. 1). Band 1 had a density of 1.127 g/ml and band II, a density of 1.142 g/ml. The occurrence in continuous gradients of two bands of particles containing rhodopsin has been noted previously (De Grip, Daemen and Bonting, 1972; Zimmerman et al., 1976; Krebs and Kuhn, 1977), but the present method of fractionation, with the sample applied in dense solution at the bottom of the gradient, provides better separation of the two bands from each other, and from denser particles, than we have bbtained before. On direct visual examination of the gradients, the two bands
SOLUBLE
PROTEINS
OF ROD
OUTER
SEGNENTS
487
appeared to be composed of finely-divided material. Examination under the phasecontrast microscope of wet preparations of samples taken from the gradients with a minimum of agitation revealed that all visible particles were dispersed; no aggregates were present. The wet preparations also showed that the particles in band II were larger than those in band I and were mostly rod-shaped, though they tended to become rounded -Density
tl
I.127 1 I
(g/ml)
I.140 I II
b---4
Fraction
I
number
FIG. 1. Isopynic sucrose gradient centrifugation of rod outer segment,s. Details are given under “Methods”. The densities shown were obtained by weighing measured volumes of the fractions indicated. The baby show fractions that were, in some cases, combined for further study. ( l ), Rhodopsin; (13) protein.
FIG. 2. Electron micrographs of the particles of band II(a) and band I(b). Particles were collected from fractions pooled as described under Fig. 1. Magnification is 9700x ; the bar indicates 1 pm.
48X
W. (:ODC.‘HAl’S
ITI
ASD
\\‘.
1’. %lIU.\~E1~21AK
perhaps through swelling or folding- -upon standiug or upon riilutioil ant1 t#ion. Figure 2(a) shows an electron micrograph of a typical field of a s&ion f’ronl t.lrcs pellet of particles from band II. The particles were large (IL10 ~111in ;;cctiou). COIItwined the membrane lamellae t,ypicwl of outer segments and were sunour~rlctl ‘1~ an intact outer (plasma) membrane. Examination of numerous fields in several secttouh failed t’o reveal the presence of significant numbers of contanlinaCing pa’ticles exterior to the outer segment plasma membranes, nor did it show thr‘ prcencch of an! cilia. basal bodies. mitochondria or nuclei within bhe structures containing clip lamellac. The outer segment’s did contain some internal vesicles that, appearecl t,o I)(> more rounded than typical disc membranes: these were devoid of internal nleml)raneq and also of densely-staining contents and may have been swollen disc meml~~nt~s. In contrast to the particles of band II, t)hose of band 1 were smaller and appesretl to be mostly isolated groups of disc membranes not surrounded by an intact plasma membrane [Fig. f?(b)]. H ere, also: occasional small rounded vesicle,!: without obviou’: internal struct,ure, were present. C~‘lltl.ifll~ik
Protein
co&at
of the
roaouter
segment frnctiom
Band I (Fig. 1) contained 44 mg protein/pmol rhodopsin (range, 42 to 46 in replicate preparations). whereas band II contained 56 mg protein/pmol rhodopsin (range, 55 to 58). Gel electrophoresis of the tot’al prohein in particles collected from the pooled gradient fractions revealed that band I contained, almost exclusively, opsin (protein 37”) and its dimer (protein 74): trimer (protein 111) and, possibly, higher aggregates [Fig. 3(a), (b)]. Th e relative amount of multimeric forms was less when the amount of protein applied to the gels was decreased. Band I also contained traces of a few other species, notably protein 47. In contrast, band II contained more than trace amounts of a number of polypeptides other than opsin [Fig. 3(c), (d)]. The major species detectable were proteins 95, 53? 50 and 41. Particles were collected from the pooled gradient fractions, and protein was extracted from the particles with hypotonic buffer (5 mM-Tris buffer). Extraction of the particles of band II yielded 30% of the protein, and less than 2% of the rhodopsin, in the supernatant, whereas extraction of the particles of band I yielded less than 2% of the protein in the supernatant (Table I). Gel electrophoresis of these extracts [Fig. 3(g)] sh owed that proteins 95, 53, 50 and 51 were present in extracts from band II, but not band I, and these gels revealed the presence in band II of an additional polypeptide, protein 375, that was obscured by the dense opsin band in the gels of the total protein. The distinction between protein 378 and opsin is discussed further, below. Gel electrophoresis of the membrane sediment obtained after extraction of the particles of band II showed that most of proteins 95, 53, 50 and 41 had been removed [Fig. 3(f)], though it has been our experience that trace amounts of those species remain in the sediment even after extensive washing. Figure 4 shows a densitometric scan of the electrophoretic pattern of the proteins extracted from the particles found in band II. The amounts of individual species (as a percentage of the total soluble proteins assessed from the areas under the curve) were: protein 375, 28%; protein 41, 19%; protein 53 plus protein 50, 14% and protein 95, Sq&. Protein 375 does not separate from opsin on gel electrophoresis in detergent solution, but the two proteins are, nonetheless, distinct. Extraction of the intact * For brevity,
“protein
37” (for example) is used to refer to a polypeptide
of apparent mol. wt. 37 000.
SOLUBLE
PROTEINS
OF ROD
OUTER
489
SEGMEKTS
50
41 37
abcdef
g
h
i
j
k
I
%‘Iort,ions. Two minor components were also observed ; one that had a density Density 3 I.049 I
(g/ml) I.096 I
I.114 I
Fraction number
FIG. 10. Isopycnic centrifugation of intact and disrupted outer segments on isoto:lic gradients. Particles were collected from band II(a) and band I(b) of the preparative sucrose gradient and centrifuged on gradients of sucrose-metrizamide as described under “Materials and Methods”.
4Yti
W. GODCHAUS
III
AND
\C. F. ZIMIMERJIAX
slightly higher than the majcr component (this. also, contained the full cotnpletnr~nt of proteins) and one at a density of approximately 1.096 g/ml that was fonntl, cn gel electrophoresis, to be virtually devoid of proteins other than opsin. This tlrnsest COIW ponent probably resulted from disruption of some of the particles during manipulxtion. The particles of band I had a buoyant density of l-096 g/ml in thr isobmic gradient [Fig. IO(b)]. 4. Discussion The most likely explanation of the above observations is that rod outer segments contain, in addition to the membrane protein rhodopsin, substantial quantities of a few specific soluble proteins, amount’ing to about 309/o of the total protein of the organelle. The procedure used here for fractionation of retinas yields some rod outer segments that are morphologically intact and that contain the soluble proteins in an osmotic compartment, and some aggregates of disc membranes from outer segments that were disrupted during manipulation and have lost their soluble proteins. Occurresaceof soluble proteins in rod outer segments There is much evidence for the physical association of the soluble proteins 95, 53, 50, 41 and 3% with intact (band II) rod outer segments. These proteins were concentrated in fractions of the isopycnic gradient where the rhodopsin of intact outer segments was concentrated, and did not separate from the rhodopsin-containing structures during zone sedimentation of the particles. The buoyant density in sucrose gradients of disrupted outer segment material (band I) is, presumably, close to that of the disc membranes themselves. The greater density of the intact outer segments (band II) is consistent with the presence, in addition to the membranes. of an RSSOciatecl osmotic compartment cont’aining the soluble proteins (and possibly other macromolecules), highly concentrated hy virtue of the exposure of the particles to hypertonic sucrose solubions. Exposure of the intact outer segments to hypotonic media, a procedure that releases the soluble proteins from a particulate component, also converts the rhodopsin-containing structures to a form having the lower buoyant density. This provides an additional correlation between the hehaviour of the soluble proteins and that of the authent)ic outer segment structures. Also2 the soluble proteins banded with the rhodopsin in both the sucrose (hypertonic) and the isotonic gradients, in which the outer segments exhibited very different buoyant densiCes (and. therefore, particle volumes). It’ seems very unlikely that an unattached contaminating part’iole would have the same density as outer segments in the two different types of gradient. It seems probable that the soluble proteins were contained within the outer scgments and not within other subcellular structures that were adventitiously attached to their surface. Estimates of the concentration of rhodopsin in outer segmcrus range from 2 to 7 mu (reviewed by Rotlieck. 1973). The content of soluble prot’eins is about 17 mg/pmol rhodopsin (Table I). so their total concentration would tJe 34 to 120 mg/ml. Because of the presence of such large quantities of t,he soluble proteins, their presence in other particles would require that these particles be present in ntore than trace quantities. Furthermore. the soluble proteins present, in intact outer segments contained a limited collection of polypeptides; attached particles containing them would, presumably, have been limited to one or a very few types. Yet electron micrographs did not reveal the presence of large numbers of particles exterior tfo the outer segment plasma membranes. It is also unlikely that the proteins as such were
SOLUBLEPROTEINS
OFROD
OUTERSEGNENTS
497
adsorbed to the external surface of the plasma membrane of the outer segments, since simple transfer to a medium without sucrose (a change in osmotic pressure but not, in ionic strength) was sufficient to solubilize the proteins. It is also probable that proteins 95, 53, 50, 41 and 375 are native to the outer segment structures that were isolated, and were not introduced from the homogenate into the interior of the outer segments during breakage of the rod cell membrane. Three of the polypeptides extracted from the intact outer segments were found only a,s very minor components of the general soluble fraction of the homogenate or of ext.racts prepared from other particles, and t,hese preparat.ions contained species not present in the outer segments. This observation rules out the random inclusion of a portion of the external medium when the outer segment plasma membra,ne was llroken and resealed. Homogenization of retinas in ten times the usual volume of medium did not alter the amount or soluble protein content of band II outer srgrnents. It is therefore unlikely that the soluble proteins entered the outer segments 1)~.tliffl~sion front other phases of the homogenate. It has previously been suggested that the high buoyant density of’ some of t’hc outer segments might reflect the presence of a port’ion of the inner segment t,hat. remains attached to those structures (McClonnell, 1965). The cilium and its basal I)ody and the ellipsoid (mitochondrial) body are the inner segment structure lying nearest to the outer segment (Young, 1969),. we have been unable to observe these &u&ares in the sections of the particles of band II. It is possible, of course, that a portion of the cytoplasm of the inner segment not including these easily-identifiable st,ructures. was retained by the outer segments. However; the rarity of structures t,ypic.al of the inner segment in the sections a,ntl hhe finding of rather large quantities of soluble. prot,ein in the particles militates against the possibility that the prot,cins are derived exclusively from the inner segment. The question of whether any soluble priteins are confined exclusively to the outer segment cannot be addressed without means for examining specifically the contents of the inner segment; how-ever, three of the soluble proteins(95, 41 and 378) may well l)e peculiar either to the outer segment or to t)he rod cell. The general soluble fract’ion of the homogenate should be representative of the cytosol proteins of the several tlifferent, cell-types of the retina. Proteins 95, 41 and 37s were present, if a,t all, as \rery minor components of this fraction; such quantities as were present could have been derived from the disrupted outer segments or from the inner segment. Similarly, t,he three polypeptides are not major components of extracts prepared from other suhcellular fractions. There was some tendency for the three proteins to he present in hmall amounts in gradient fractions denser t)han band II (most noticeable for a l)rotein that may be 41, Fig. 6). This may have resulted from breakage of sonle outer segments-with deposition of their soluble probein--during passage through the gradient, or from the presence of small amounts of outer segments (difficult to detect a,t low concentration) in denser fractions. The former view is supported by the ohservation that the st)aining density of the protein hands decreased more with increasing density when extracts from particles collected by centrifugation (and thus freetl of extraneous soluble proteins) were examined (Fig. 7). Osmotic beheior
of the outer segments
The results obtained in this study strongly suggest that proteins 95, 53, 50: 41 ant1 378 are locat,ed in one or more solution phases confined within osmotic compartments
40X
I\:.
GOD(‘HAYS
III
AA-l)
\V. I’. %IMJlE1!~.\1i\S
that are present in the intact outer segrnent,s. disrupted by osmotic shock. at1~1alJs(bnt from the discs found in band I. There are other possible explanations, for cxutt~~~lc. that the proteins might be adsorbed to a membrane and released when the nlcn tl)rane is disrupted and the complex is exposed to media of low ionic st,rength. It is clificrtlt to understand how all five different polypept,ides could exhibit, the same wtlsorpticJt1 properties, however. Studies of the osmotic behavior of the outer segment fract’ions provide evidence for the existence of one or more osmotic compartments that are present in 6he int’act outer segments but not in the isolated discs. In the sucrose gradient, the intact, outer segments came to isopycnic equilibrium at a density of 1.142 g/ml, whereas their density in the isotonic gradient was 1.049 g/ml. If it is assunl& that the densit,) change resulted only from uptake of water, with no loss of solutes, then the volume of the particles in the solution of physiological osrnolarity was 190”,, greater than that in the 1.3 osmolar sucrose solution. The conclusion that such a change in volume took place is supported by the microscopic observations of t,he particles in solutions of the same osmolarities (Fig. 9) ; the average diameter of the particles in the solution of low osmolarity appears to be at least 1.5 times that in t*he solution of high osmolarity. In contrast, the discs (band I) had a density of 1.125 in the sucrose gradient (where they banded at an osmolarity of 1.14) and a tlensity of 1.096 in the gradient that was of physiological osmolarity. This corresponds to a volume increase of 30%; even if this is corrected to 35‘:,(,to account for the difference in the osmolarities at which the intact outer segments and the discs band in t(he sucrose gradient, it is small compared t#o the volume change of the intact. particles. The intact outer segments, therefore, contain one or more osmotic compartments that can be distinguished from the compartment within the discs themselves on the basis of behavior in solutiolis of clifferent osmolarity. The very small proporLiona1 increase in the volume of the discs between external osmolarities of l-14 and 0.32 would be explained if the disc vesicles contained a very small amount of solutes that contribute to the internal osmotic pressure; the discs lvould then be in virtually collapsed form at both these external osmolarities, as they appear to he in vivo (Young, 1969) where the external osmotic pressure should not exceed the physiological level. In fact, the discs can he observed to swell much more dramatically when transferred from 0.32 M-sucrose to the 5 mNl-Tris buffer without sucrose (assessed by measuring the volume of their pellet after centrifugation; unpublished results of the authors). This behavior is consistent with the ahove i&rpretation. Membranes obtained after extraction of the solul,le proteins from the intact outer segments with the 5 m;M-Tris buffer could still be observed to swell and shrink reversibly. Their internal compartment may therefore remain intact even during exposure to very low external osmolarities; presumably, the paucity of internal solutes results in an internal pressure insufficient to cause rupture of the membrane. For this reason, it seems unlikely that the proteins that were extracted by the dilute buffer were contained within the internal compartment of the discs themselves. Confinement of the soluble proteins within only the plasma membrane is sufficient to account for their behavior, but it is possible that yet a third osmotic compartment within the plasma membrane might contain the soluble proteins. Conceivably, the rounded vesicle observed in electron micrographs could play such a role, though these did not have densely-stained contents and also appeared to be present in the material from band I, that was essentially devoid of soluble proteins. If the compartment within the plasma membrane is, in fact, intact in the particles of band II, it must be
SOLUBLE PROTEIKS
OF ROD OUTER SEGMENTS
499
supposed either that the plasma membrane reseals after separation of the outer segment from the inner segment during disruption of the tissue, or else that there is a barrier between the two segments in vivo. Implications
for the physiological
study of outer segments and rhodopske
The integrity of isolated rod outer segments is of great importance to the study of their function in vitro. The methods described here provide a means of separating intact from disrupted outer segments and provide three criteria of integrity that are independent of microscopy : buoyant density, osmotic behavior and content of certain specific proteins. Purity of the preparations is equally important. The intact outer segment fraction, as prepared here, contains small amounts of proteins-most of them not extractable by hypotonic buffer-that show no correlation wyith rhodopsin in the density gradient fractions (e.g. protein 103). It must be suspected, at least, that these are contaminants. Assays for enzyme activities should be carried out on gradient fractions similar to those described here, to determine whether particular activities are in fact native to the outer segment. The re.sults of this study are consistent with the generally-accepted view that. rhodopsin constitutes over 90% of the membrane protein of outer segments (Heitzmann, 1972). However, gel electrophoresis in solution of strong detergents is currently t,he only practical means for assessing the freedom of rhodopsin preparations from small amounts of other proteins. Protein 37% and small amounts of protein 41 would he obscured by the opsin band in these gels (Fig. 4). These observations indicate the need for a cautious approach to the assumption that even well-washed membranes contain only a single protein, rhodopsin. Outer segment proteins are not synthesized locally, but. are transported from the inner segment (Young, 1974). We have recently investigated the incorporation of radioactive amino acids into protein in a cell-free system from bovine retina (Godchaux, 1978). The polypeptides synthesized by that system included (among others) species of molecular weight (15%) 50 000, 47 000, 41 000 and 36 000 (the system wa,s probably not active enough to complete many chains of mol. wt. 95 000). These four species accumulated at very high specific activity, and as completed chains no longer associated with transfer RNA, in particles that sedimented with the microsomal fraction. Three of the species (47, 41 and 36) were precipitated hy an antibody prepared against disc membrane protein that was undoubtedly contaminated with some of the soluble proteins described here [cf. Fig. 5(f)]. The labelled species of nominal mol. wt. weight 36 000 could have been opsin, protein 375, or a mixture of the two. This raises the interesting possibility, as a subject for future investigation, that some of the soluble proteins described in the present communication might be transported specifically to the outer segment and might be confined to a particulate compartment during that process.
ACKNOWLEDGMENTS The authors wish to thank Mr Glen Winkel for his excellent assistance in electron microscopy and Dr W. E. Robinson for suggesting the experiments with isotonic gradients. This work was supported by a grant (EY-00675) to W. F. Z. from the National Institutes of Health and by Institutional Funds (assigned to W. G.) granted to Amherst College by that agency.
REFERENCES Bownds, D. and Brodie, A. E. (1975). Light-sensit,ive swelling of isolated frog rod outer segme~~th as an in vitro assay for visual transduction and dark adaptation. J. (/e?b. Physiol. 66, 41)7 -S.i. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantit.iw of protein utilizing the principle of’ protein-dye binding. Analyt. Biochem. 72, 248~-54. De Grip, W. J., Daemen. F. J. M. and Bont)ing, S. L. (1972). Biorhemical aspects of the visu;~l process, XVIII. Enrichment of rhodopsin in rod outer segment membrane preparation. Vision Rev. 12, 1697-707. Frank. R. N. and Buzney, S. M. (1975). Mechanism and specificit,y of rhodopsin phosphorylat’ion. Biochem,istry 14, 5110-17. Godchaux, W. (1978). Biosynthesis of proteins in a cell-free syst,em from bovine retina. Bioche,,/. Biophys. Acta Ko. 99232, in the press. Hagins, W. A. and Yoshikami, S. (1974). A role for calcium ion in excitat’ion of retinal rods and cones. Exp. Eye Res. l&299-305. Hemminki, K. (1975). Accumulation of calcium by retina,1 outer segments. Bctrc Physiol. &and. 95, 117-25. Heitzmann, H. (1972). Rhodopsin is the predominant protein of rod outer segment membranes. Nature, New Biol. 235, 114. Jan, 1,. 1’. and Revel, J. (1974). Ultrastructural localization of rhodopsin in vertebrate retina. J. Cell Biol. 62, 257-72. Krebs, W. and Kuhn, H. (1977). Structure of isolated bovine rod outer segment membranox. Exp. Eye Res. 25,511-26. Kuhn, H., Cook, J. H. and Dreyer, W. J. (1973). Phosphorylation of rhodopsin in bovine photoreceptor membranes, a dark reaction after illumination. Biochemistry 12, 2495-502. Lowry, 0. H., Rosebrough, N. J., Parr, A. L. and Randall, R. J. (1951). Protein measurements with Polin phenol reagent. J. Biol. Chem. 193, 265-75. Maizel, J. V., Jr. (1971). Electrophoresis of viral proteins. Methods in Virology 5,180-247. McConnell, D. G. (1965). The isolat’ion of retinal outer segment fragments. J. Cell, Biol. 27,459-73. Miki, N.. Baraban, J. M., Kearns, J. J., Boyce, J. J. and Bitenski, M. W. (1975). Purification and properties of the light-activated phosphodiesterase of rod out,er segments. J. Biol. C~PYYI. 250,6320-27. Papermaster, D. S. and Dryer, W. J. (1974). Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 13, 243844. Papermaster, D. S., Converse, C. A. and Siu, J. (1975). Membrane biosynthesis in the frog retina: Opsin transport in the photoreceptor cell. Biochemistry 14, 1343-52. Rickwood, D. and Birnie, G. D. (1975). Metrizamide, a new density-gradient medium. Frd. F:ur. Biochem. Sot. Letters 50, 102-10. Rodieck, R. W. (1973). The Vertebrate Retina: Principles of Xtructure and Function. Pp. I 11-12. W. H. Freeman and Co., San Francisco. Saari, J. C. and Futterman, S. (1976). Retinol-binding protein in bovine retina: Isolation and partial characterization. Exp. Eye Res. 22, 425-35. Young, R. W. (1969). The organization of vertebrate photoreceptor cells. In The Retina: MorphoEogy, Function and Clinical Characteristics (Eds Straatsma, B. R., Hall, M. O., Allen, R. A. and Crescitelli, F.). Pp. 177-210. University of California Press, Berkeley. Young, R. W. (1974). Biogenesis and renewal of visual cell outer segment membranes. Exp. Eye Res. 18, 215-23. Zimmerman, W. F., Lion, F., Daemen, F. J. M. and Bonting, S. L. (1975). Distribution ofstereospecific retinol dehydrogenase activities in subcellular fractions of retina and pigment epithelium. Exp. Eye Res. 21, 325-32. Zimmerman, W. F., Daemen, F. J. M. and Bonting, S. L. (1976). Distribution of enzyme activities in subcellular fractions of bovine retina. J. Bill. Chem. 251, 4700-5.
