Exp. Eye Res. (1992) 54, 821-823
LETTER
Phospholipid
TO THE
Distribution Among Bovine Rod Outer Plasma Membrane and Disk Membranes
Retinal rod cell disks arise from evaginations of the plasma membrane at the base of the rod outer segment (ROS). Upon the formation of a disk membrane from the ROS plasma membrane the lipid and protein components must be sorted to achieve a new independent membrane. In this report we compare the phospholipid headgroup composition of the plasma membrane to that of the disk membranes. Previously we showed that the ROS plasma membrane has a cholesterol to phospholipid mole ratio of 0.38. The cholesterol to phospholipid ratio of disks, however, ranges from 0.32 in the basal membranes to 0.05 in the apical membranes (Boesze-Battaglia, Hennessey and Albert. 1989). The total fatty acid composition of the plasma membrane is distinctly different from that seen in newly formed basal disks (Boesze-Battaglia and Albert, 1989). ROS disk and plasma membranes were isolated from the same preparation of frozen bovine retinas (J. Lawson, Inc., NE). Plasma membrane was isolated by exploiting its ability to bind ricin as described (Molday and Molday, 198 7) and modified (Boesze-Battaglia and Albert, 1989). Molday and Molday (1987) have demonstrated that these binding sites are located exclusively on the surface of the ROS plasma membrane. Plasma membrane isolated using this technique has a fatty acid composition distinct from that of disks (Boesze-Battaglia and Albert, 1989). SDS-PAGE was also used to compare the disk and plasma membrane fractions. As previously observed by Molday and Molday, (1987). proteins of molecular weight 63 000 and 240000 Da were observed in the plasma membrane fraction but not in the disks. The most intensely Coomasie stained band in both membrane preparations corresponds to a molecular weight of 40000 Da (rhodopsin). ROS disk and plasma membrane lipids were extracted as described by Polch, Lees and Sloane-Stanley ( 19 5 7). The lipids were isolated from the lower organic phase, dried under nitrogen and then resuspended in hexane. The phospholipid headgroup composition of these total lipid extracts was immediately determined using high-pressure liquid chromatography (HPLC) on a LiChrosorb SI-60 (silica gel column) as described previously (Hax and Van Kessel, 19 77). The phospholipids were identified by comparison to known standards (Avanti Polar Lipids). The collected fractions were also subjected to thin-layer chromatography (TLC) as described below to assessthe purity of the eluted peak. The individual lipid peaks were collected, dried down 0014-4835/92/050821+03
EDITORS
$03.00/O
Segment
and phosphate concentrations determined as described below. The total lipid extracts were also analysed for phospholipid headgroup composition using twodimensional TLC (Kates, 1975). The phospholipids were detected and identified using Ninhydrin (for amino phospholipids). Dragendorf (for choline phospholipids) and sulfuric acid (for total composition) staining procedures as described (Kates, 1975). The phospholipids were identified by comparison to the migration of known phospholipid standards (Avanti Polar Lipids). Sample spots were scraped and phosphate was determined by the method of Bartlett (19 59) as modiied by Litman (19 73). Cholesterol was determined as described by Allain et al. ( 19 74). The phospholipid headgroup composition of the plasma membrane and of the disk membranes isolated from the same sucrose density gradients are presented in Table I. The values presented are an average of HPLC and TLC runs of three independent preparations of disks and plasma membrane. The values given represent the mole percent of the total phosphate recovered. Several differences in phospholipid headgroup composition between the disk and plasma membranes are apparent. In the plasma membrane phosphatidylethanolamine (PE) accounts for only 11% of the total phospholipid, In the disk membranes PE represents greater than 40% of the total phospholipid. Phosphatidylserine (PS) is present in the plasma membrane (24%) at twice the level found in the disk membranes. Phosphatidylcholine, which constitutes approximately 40% of the disk membrane phospholipid, accounts for 65% of the phospholipid in the plasma membrane, In previous determinations of the phospholipid headgroup composition of the disk membranes, PE represents approximately 32 %, phosphatidylcholine (PC) 35%. PS 12 % and phosphatidylinositol (PI), l-2% of the total phospholipid (Fliesler and Anderson, 1983). Our results agree well with these determinations. It has previously been shown that there is also a remarkable difference in the fatty acyl components of the membrane phospholipids between disks and the surrounding plasma membrane. The ROS plasma membraneishighin18:1,and18:2andlowin22:6 when compared to the disk membrane. The ROS plasma membrane has also been shown to have a cholesterol to phospholipid mole ratio of 0.3 8 (BoeszeBattaglia and Albert, 1989). Disks are progressively displaced toward the apical 0 1992 Academic Press Limited
822
K. BOESZE-BATTAGLIA TABLE
1
Phospholipid headgroup composition of ROS membranes Phospholipid
Disk membrane
Plasma membrane
PE
41.6+2.6*
PS PI
13.7k2.1 2.5 10.8 45.3 -+_3.2
10.6k2.8 24.1& 2.8 < I.0 65.1 &- 3.8
PC
* Data are represented as mole percent of total phosphate.
t 0
I
I 0.1
P A-
Cholesterol/phospholipid
T
I
I
0.2 mole
I
0.3 ratio
FIG. 1. Mole percent phospholipidin bovine ROS disk membranes as a function of membrane cholesterol/phospholipidratio. Disk membraneswere separatedon the basisof cholesterolcontent, which is reflectiveof the ageand spatial distribution of these membranes.The phospholipid headgroup composition of disk subpopulationswas determined by HPLC and TLC as describedin the text. The phospholipidrecoveredin each HPLC peak and TLC spot correspondingto PC(+), PE(a), PS( x ) or PI (0) isshown as a percentageof the total phospholipid.The data shown are an average of four determinationson two separate preparations.Of the four determinations,two were HPLC and two wereTLC. Error barsrepresent&s.D. and are given for all phosphatevalues.
tip of the outer segment as additional disks are formed. During this processof apical displacement the membrane protein associatedwith a disk does not undergo turnover (Young, 1976). The membrane phospholipids, however, do undergo rapid metabolic turnover (Anderson and Maude, 1970; Bibb and Young, 1974a.b: Anderson, Kelleher and Maude, 1980: Anderson et al., 1980 ; Anderson, Maude and Kelleher, 1980). We have shown that the lipid composition of the plasma membrane is distinct from that observed for a preparation of total disk membranes. The question which then arises is whether the disk lipid composition is modified during the time of disk transit from the outer segment base to the apical tip. Therefore, disk lipid composition was also characterized as a function of disk age and spatial location. We have previously shown that disk membranes can be separated into subpopulations which differ in membrane cholesterol content (Boesze-Battaglia, Hennessey and Albert, 1989). The cholesterol to phospholipid mole ratio of the disk membranes
AND
A. D.ALBERT
decreaseswith disk age and hence reflects the spatial location of the disks in the outer segment (BoeszeBattaglia, Fliesler and Albert. 1990). In this report the separation basedon disk cholesterol content is used to investigate the phospholipid headgroup composition of disk membranes as a function of their spatial displacement in the ROS. ROSdisk membranes were separated into subpopulations which vary in membrane cholesterol to phospholipid mole ratios from 0.26 to 0.06. (The maximum range of cholesterol to phospholipid ratios is not apparent because the subpopulations were pooled to achieve sufficient amounts of phospholipid for accurate analysis.) The phospholipid headgroup composition of each disk membrane subpopulation was then determined. The major phospholipids, PC, PE, PS and PI were identified using two independent techniques: HPLC and TLC as described above. In Fig. 1 the mole percent of the total phosphate is plotted as a function of the cholesterol to phospholipid ratio of the disk membranes. Figure 1 shows the phospholipid composition of disks as a function of their spatial location in the ROS with the oldest disks exhibiting the lowest cholesterol to phospholipid ratio and the newest disksexhibiting the highest cholesterol to phospholipid ratio. As can be seen in Fig. 1 the phospholipid headgroup composition of the disk membranes, separated on the basis of cholesterol content., does not change as a function of the cholesterol to phospholipid ratio. PE and PC make up approximately 41 and 44x, respectively, of the total phospholipid in the disks. The PS and PI content of the disks is approximately 14.5 and l-2%, respectively. The phospholipid composition of control disk membranes (which were not treated with digitonin) is PC 3&O%, PE 45%. PS 13.5% and PI 2.6% (data not shown). These values are in good agreement with previous determinations of the total phospholipid composition of disk membranes (Fliesler and Anderson, 1983). It was shown previously in this laboratory that there are no changes in the total fatty acid composition of the disks as a function of age (Boesze-Battaglia et al., 1989). Data presented here indicate that the phospholipid composition of the plasma membrane is distinctly different from that of the disks. However, the phospholipid composition of the disks does not undergo subsequent change as the disks are apically displaced even though the disk phospholipids undergo turnover. The difference in phospholipid composition between the disk membranes and the plasma membrane may be essential to establish the cholesterol distribution found in the ROS. Previous results on cholesterol equilibration between disks and vesicles suggest that the cholesterol distribution is governed by the phospholipid composition of the membranes (House. Badgett and Albert, 1989). Model studies have shown that cholesterol will partition out of a membrane high in PE into a membrane with high levels of PC (Yeagle
PHOSPHOLIPID
DISTRIBUTION
IN PLASMA
AND
DISK
and Young, 7 986). The ROS plasma membrane is lower in PE and higher in PC than are the disk membranes. Thus, it is expected that the apparent cholesterol equilibrium will favor the plasma membrane over the disk membranes. It is tempting to speculate that the cholesterol heterogeneity in the disk membranes is due to a temporal partitioning of disk membrane cholesterol into the surrounding plasma membrane. It has also been shown that cholesterol can partition between blood plasma lipid carrier proteins and rod outer segments (Steck, Kezdy and Lange, 1988). Thus, the final cholesterol content in the disks may reflect an apparent equilibrium between the disks, ROS plasma membrane and lipid carrier proteins. These data are consistent with a mechanism of lipid sorting which regulates the phospholipid composition of disks as they form. The cholesterol content of disks is in turn governed by the phospholipid composition, leading to a time-dependent flux of cholesterol out of the disk membranes. Membrane cholesterol composition has important functional consequences for the rod cell. Rhodopsin is the major membrane protein in both the disk and plasma membrane. Previous studies have shown that upon absorption of a light, the rhodopsin in the plasma membrane will not activate the cGMP cascade as readily as rhodopsin in the disk membranes primarily due to the high cholesterol environment of the plasma membrane relative to the disk membranes (Boesze-Battaglia and Albert. 1990). This inhibitory effect is removed when the plasma membrane cholesterol level is decreased. Together these data suggest an elegant mechanism whereby the phospholipid sorting determines the membrane cholesterol level which in turn modulates the membrane receptor activity. Acknowledgements This work was supported by grants from the National Eye Institute (EYO3 328 to A. D, A. and EYO6241 to K.B. B.). The authors would like to thank M. Haldeman for some of the preliminary work in this study. KATHLEEN BOESZE-BATTAGLIA ARLENE D. ALBERT Departments of Biochemistry and Opthalmology, University at Buffalo School of Medicine (SUNY), Buffalo, NY 74274, U.S.A.
References Allain. C. C., Poon. L. S., Chan. C. S. G.. Richmond,W. and Fu. P. C. (1974). Enzymatic determination of total serumcholesterol.Clin. Chem.20. 470-S.
(Received
Rockville
21 November
1991
823
MEMBRANES
Anderson, R. E.. Kelleher, P. A. and Maude, M. B. (1980). Metabolism of phosphatidylethanolamine in the frog retina. Biuchim. Biophys. Actn 620. 227-35. Anderson, R. E., Kelleher, P. A., Maude, M. B. and Maida, T. M. (1980). Synthesis and turnover of lipid and protein components of frog retinal rod outer segments. Neurochemistry 1, 2942. Anderson, R. E. and Maude, M. B. (1970). Phospholipids of bovine rod outer segment. Biochemislry, 9, 3624-8. Anderson, R. E.. Maude. M. B. and Keileher. P. A. (1980). Metabolism of phosphatidylinositol in the frog retina. Hiochim. Biophys. Acta 620, 23646. Bartlett, C. R. (1959). Phosphorus assay in column chromatography. j. Biol. Chem. 234, 466-73. Bibb, C. and Young, R. W. (1974a). Renewal of fatty acids in membranes of visual cell outer segments. 1. Cell Biol. 61, 327-43. Bibb, C. and Young, R. W. (lY74b). Renewal of glycerol in visual cells and pigment epithelium of the frog retina. J. Cell Biol. 62. 378-89. Boesze-Battaglia, K. and Albert, A. D. (1989). Fatty acid composition of bovine rod outer segment plasma membrane. Elcp. Ege Res. 49, 699-701. Boesze-Battaglia, K. and Albert, A. (1990). Cholesterol modulation of photoreceptor function in bovine rod outer segments. J. Biol. Chem. 265. 20727-30. Boesze-Battaglia. K.. Fliesler S. J. and Albert, A. D. (1990). Relationship of cholesterol content to spatial distribution and age of disk membranes in retinal rod outer segments. 1. Biol. Chem. 265. 18867-70. Boesze-Battaglia. K., Hennessey, T. and Albert, A. D. (1989). Cholesterol heterogeneity in bovine rod outer segment disk membranes. 1. Biol. Chem. 264. 8151-S. Fliesler. S. J. and Anderson, A. E. (1983). Chemistry and metabolism of lipids in the verbrate retina. Prog. Lipid Res. 22. 79-131. Folch,J., LeesM. andSloane-Stanley.G. A. (1957). A simple methodfor the isolationand purification of total lipids. I. Biol. Chem.226, 497-509. Hax, W. M. and Van Kessel, W. S.M. ( 1977). Highperformance liquid chromatographic separation and photometric detection of phospholipids.J. Chromatography 142, 73541. House,K.. Badgett,D. and Albert, A. D. (1989). Cholesterol movement between bovine rod outer segmentdisk membranesandphospholipidvesicles.Exp. EyeRes.49. 561-72. Kates,M. (1975). Techniques oj Lipidology.AmericanElsevier Publishing:New York, NY. Litman. B. J. (1973). Lipid model membrane characterization of mixed phospholipid vesicles. Biochemistry 13. 2545-54. Molday, R. and Molday, L. (1987). Differencesin the protein compositionof bovine retinal rod outer segmentdisk andplasmamembraneisolatedby a ricin-gold-dextran density perturbation method. J. Cell Biol. 105, 2589-601. Steck, T., Kezdy, F. and Lange, Y. (1988). An activationco\lision mechanismfor cholesteroltransfer between membranes.1. Biol. Chem.263, 13023-31. Yeagle.P. L. and Young, J. (1986). Factorscontributing to the distribution of cholesterol among phospholipid vesicles.J. Biol. Chem.261, 8175-81. Young. R. W. (1976). Visual cells and the concept of renewal. J. Cdl. Biol. 39, 169-84.
and accepted
in revised
form
27 January
7992)
LETTER
Phospholipid
TO THE
Distribution Among Bovine Rod Outer Plasma Membrane and Disk Membranes
Retinal rod cell disks arise from evaginations of the plasma membrane at the base of the rod outer segment (ROS). Upon the formation of a disk membrane from the ROS plasma membrane the lipid and protein components must be sorted to achieve a new independent membrane. In this report we compare the phospholipid headgroup composition of the plasma membrane to that of the disk membranes. Previously we showed that the ROS plasma membrane has a cholesterol to phospholipid mole ratio of 0.38. The cholesterol to phospholipid ratio of disks, however, ranges from 0.32 in the basal membranes to 0.05 in the apical membranes (Boesze-Battaglia, Hennessey and Albert. 1989). The total fatty acid composition of the plasma membrane is distinctly different from that seen in newly formed basal disks (Boesze-Battaglia and Albert, 1989). ROS disk and plasma membranes were isolated from the same preparation of frozen bovine retinas (J. Lawson, Inc., NE). Plasma membrane was isolated by exploiting its ability to bind ricin as described (Molday and Molday, 198 7) and modified (Boesze-Battaglia and Albert, 1989). Molday and Molday (1987) have demonstrated that these binding sites are located exclusively on the surface of the ROS plasma membrane. Plasma membrane isolated using this technique has a fatty acid composition distinct from that of disks (Boesze-Battaglia and Albert, 1989). SDS-PAGE was also used to compare the disk and plasma membrane fractions. As previously observed by Molday and Molday, (1987). proteins of molecular weight 63 000 and 240000 Da were observed in the plasma membrane fraction but not in the disks. The most intensely Coomasie stained band in both membrane preparations corresponds to a molecular weight of 40000 Da (rhodopsin). ROS disk and plasma membrane lipids were extracted as described by Polch, Lees and Sloane-Stanley ( 19 5 7). The lipids were isolated from the lower organic phase, dried under nitrogen and then resuspended in hexane. The phospholipid headgroup composition of these total lipid extracts was immediately determined using high-pressure liquid chromatography (HPLC) on a LiChrosorb SI-60 (silica gel column) as described previously (Hax and Van Kessel, 19 77). The phospholipids were identified by comparison to known standards (Avanti Polar Lipids). The collected fractions were also subjected to thin-layer chromatography (TLC) as described below to assessthe purity of the eluted peak. The individual lipid peaks were collected, dried down 0014-4835/92/050821+03
EDITORS
$03.00/O
Segment
and phosphate concentrations determined as described below. The total lipid extracts were also analysed for phospholipid headgroup composition using twodimensional TLC (Kates, 1975). The phospholipids were detected and identified using Ninhydrin (for amino phospholipids). Dragendorf (for choline phospholipids) and sulfuric acid (for total composition) staining procedures as described (Kates, 1975). The phospholipids were identified by comparison to the migration of known phospholipid standards (Avanti Polar Lipids). Sample spots were scraped and phosphate was determined by the method of Bartlett (19 59) as modiied by Litman (19 73). Cholesterol was determined as described by Allain et al. ( 19 74). The phospholipid headgroup composition of the plasma membrane and of the disk membranes isolated from the same sucrose density gradients are presented in Table I. The values presented are an average of HPLC and TLC runs of three independent preparations of disks and plasma membrane. The values given represent the mole percent of the total phosphate recovered. Several differences in phospholipid headgroup composition between the disk and plasma membranes are apparent. In the plasma membrane phosphatidylethanolamine (PE) accounts for only 11% of the total phospholipid, In the disk membranes PE represents greater than 40% of the total phospholipid. Phosphatidylserine (PS) is present in the plasma membrane (24%) at twice the level found in the disk membranes. Phosphatidylcholine, which constitutes approximately 40% of the disk membrane phospholipid, accounts for 65% of the phospholipid in the plasma membrane, In previous determinations of the phospholipid headgroup composition of the disk membranes, PE represents approximately 32 %, phosphatidylcholine (PC) 35%. PS 12 % and phosphatidylinositol (PI), l-2% of the total phospholipid (Fliesler and Anderson, 1983). Our results agree well with these determinations. It has previously been shown that there is also a remarkable difference in the fatty acyl components of the membrane phospholipids between disks and the surrounding plasma membrane. The ROS plasma membraneishighin18:1,and18:2andlowin22:6 when compared to the disk membrane. The ROS plasma membrane has also been shown to have a cholesterol to phospholipid mole ratio of 0.3 8 (BoeszeBattaglia and Albert, 1989). Disks are progressively displaced toward the apical 0 1992 Academic Press Limited
822
K. BOESZE-BATTAGLIA TABLE
1
Phospholipid headgroup composition of ROS membranes Phospholipid
Disk membrane
Plasma membrane
PE
41.6+2.6*
PS PI
13.7k2.1 2.5 10.8 45.3 -+_3.2
10.6k2.8 24.1& 2.8 < I.0 65.1 &- 3.8
PC
* Data are represented as mole percent of total phosphate.
t 0
I
I 0.1
P A-
Cholesterol/phospholipid
T
I
I
0.2 mole
I
0.3 ratio
FIG. 1. Mole percent phospholipidin bovine ROS disk membranes as a function of membrane cholesterol/phospholipidratio. Disk membraneswere separatedon the basisof cholesterolcontent, which is reflectiveof the ageand spatial distribution of these membranes.The phospholipid headgroup composition of disk subpopulationswas determined by HPLC and TLC as describedin the text. The phospholipidrecoveredin each HPLC peak and TLC spot correspondingto PC(+), PE(a), PS( x ) or PI (0) isshown as a percentageof the total phospholipid.The data shown are an average of four determinationson two separate preparations.Of the four determinations,two were HPLC and two wereTLC. Error barsrepresent&s.D. and are given for all phosphatevalues.
tip of the outer segment as additional disks are formed. During this processof apical displacement the membrane protein associatedwith a disk does not undergo turnover (Young, 1976). The membrane phospholipids, however, do undergo rapid metabolic turnover (Anderson and Maude, 1970; Bibb and Young, 1974a.b: Anderson, Kelleher and Maude, 1980: Anderson et al., 1980 ; Anderson, Maude and Kelleher, 1980). We have shown that the lipid composition of the plasma membrane is distinct from that observed for a preparation of total disk membranes. The question which then arises is whether the disk lipid composition is modified during the time of disk transit from the outer segment base to the apical tip. Therefore, disk lipid composition was also characterized as a function of disk age and spatial location. We have previously shown that disk membranes can be separated into subpopulations which differ in membrane cholesterol content (Boesze-Battaglia, Hennessey and Albert, 1989). The cholesterol to phospholipid mole ratio of the disk membranes
AND
A. D.ALBERT
decreaseswith disk age and hence reflects the spatial location of the disks in the outer segment (BoeszeBattaglia, Fliesler and Albert. 1990). In this report the separation basedon disk cholesterol content is used to investigate the phospholipid headgroup composition of disk membranes as a function of their spatial displacement in the ROS. ROSdisk membranes were separated into subpopulations which vary in membrane cholesterol to phospholipid mole ratios from 0.26 to 0.06. (The maximum range of cholesterol to phospholipid ratios is not apparent because the subpopulations were pooled to achieve sufficient amounts of phospholipid for accurate analysis.) The phospholipid headgroup composition of each disk membrane subpopulation was then determined. The major phospholipids, PC, PE, PS and PI were identified using two independent techniques: HPLC and TLC as described above. In Fig. 1 the mole percent of the total phosphate is plotted as a function of the cholesterol to phospholipid ratio of the disk membranes. Figure 1 shows the phospholipid composition of disks as a function of their spatial location in the ROS with the oldest disks exhibiting the lowest cholesterol to phospholipid ratio and the newest disksexhibiting the highest cholesterol to phospholipid ratio. As can be seen in Fig. 1 the phospholipid headgroup composition of the disk membranes, separated on the basis of cholesterol content., does not change as a function of the cholesterol to phospholipid ratio. PE and PC make up approximately 41 and 44x, respectively, of the total phospholipid in the disks. The PS and PI content of the disks is approximately 14.5 and l-2%, respectively. The phospholipid composition of control disk membranes (which were not treated with digitonin) is PC 3&O%, PE 45%. PS 13.5% and PI 2.6% (data not shown). These values are in good agreement with previous determinations of the total phospholipid composition of disk membranes (Fliesler and Anderson, 1983). It was shown previously in this laboratory that there are no changes in the total fatty acid composition of the disks as a function of age (Boesze-Battaglia et al., 1989). Data presented here indicate that the phospholipid composition of the plasma membrane is distinctly different from that of the disks. However, the phospholipid composition of the disks does not undergo subsequent change as the disks are apically displaced even though the disk phospholipids undergo turnover. The difference in phospholipid composition between the disk membranes and the plasma membrane may be essential to establish the cholesterol distribution found in the ROS. Previous results on cholesterol equilibration between disks and vesicles suggest that the cholesterol distribution is governed by the phospholipid composition of the membranes (House. Badgett and Albert, 1989). Model studies have shown that cholesterol will partition out of a membrane high in PE into a membrane with high levels of PC (Yeagle
PHOSPHOLIPID
DISTRIBUTION
IN PLASMA
AND
DISK
and Young, 7 986). The ROS plasma membrane is lower in PE and higher in PC than are the disk membranes. Thus, it is expected that the apparent cholesterol equilibrium will favor the plasma membrane over the disk membranes. It is tempting to speculate that the cholesterol heterogeneity in the disk membranes is due to a temporal partitioning of disk membrane cholesterol into the surrounding plasma membrane. It has also been shown that cholesterol can partition between blood plasma lipid carrier proteins and rod outer segments (Steck, Kezdy and Lange, 1988). Thus, the final cholesterol content in the disks may reflect an apparent equilibrium between the disks, ROS plasma membrane and lipid carrier proteins. These data are consistent with a mechanism of lipid sorting which regulates the phospholipid composition of disks as they form. The cholesterol content of disks is in turn governed by the phospholipid composition, leading to a time-dependent flux of cholesterol out of the disk membranes. Membrane cholesterol composition has important functional consequences for the rod cell. Rhodopsin is the major membrane protein in both the disk and plasma membrane. Previous studies have shown that upon absorption of a light, the rhodopsin in the plasma membrane will not activate the cGMP cascade as readily as rhodopsin in the disk membranes primarily due to the high cholesterol environment of the plasma membrane relative to the disk membranes (Boesze-Battaglia and Albert. 1990). This inhibitory effect is removed when the plasma membrane cholesterol level is decreased. Together these data suggest an elegant mechanism whereby the phospholipid sorting determines the membrane cholesterol level which in turn modulates the membrane receptor activity. Acknowledgements This work was supported by grants from the National Eye Institute (EYO3 328 to A. D, A. and EYO6241 to K.B. B.). The authors would like to thank M. Haldeman for some of the preliminary work in this study. KATHLEEN BOESZE-BATTAGLIA ARLENE D. ALBERT Departments of Biochemistry and Opthalmology, University at Buffalo School of Medicine (SUNY), Buffalo, NY 74274, U.S.A.
References Allain. C. C., Poon. L. S., Chan. C. S. G.. Richmond,W. and Fu. P. C. (1974). Enzymatic determination of total serumcholesterol.Clin. Chem.20. 470-S.
(Received
Rockville
21 November
1991
823
MEMBRANES
Anderson, R. E.. Kelleher, P. A. and Maude, M. B. (1980). Metabolism of phosphatidylethanolamine in the frog retina. Biuchim. Biophys. Actn 620. 227-35. Anderson, R. E., Kelleher, P. A., Maude, M. B. and Maida, T. M. (1980). Synthesis and turnover of lipid and protein components of frog retinal rod outer segments. Neurochemistry 1, 2942. Anderson, R. E. and Maude, M. B. (1970). Phospholipids of bovine rod outer segment. Biochemislry, 9, 3624-8. Anderson, R. E.. Maude. M. B. and Keileher. P. A. (1980). Metabolism of phosphatidylinositol in the frog retina. Hiochim. Biophys. Acta 620, 23646. Bartlett, C. R. (1959). Phosphorus assay in column chromatography. j. Biol. Chem. 234, 466-73. Bibb, C. and Young, R. W. (1974a). Renewal of fatty acids in membranes of visual cell outer segments. 1. Cell Biol. 61, 327-43. Bibb, C. and Young, R. W. (lY74b). Renewal of glycerol in visual cells and pigment epithelium of the frog retina. J. Cell Biol. 62. 378-89. Boesze-Battaglia, K. and Albert, A. D. (1989). Fatty acid composition of bovine rod outer segment plasma membrane. Elcp. Ege Res. 49, 699-701. Boesze-Battaglia, K. and Albert, A. (1990). Cholesterol modulation of photoreceptor function in bovine rod outer segments. J. Biol. Chem. 265. 20727-30. Boesze-Battaglia. K.. Fliesler S. J. and Albert, A. D. (1990). Relationship of cholesterol content to spatial distribution and age of disk membranes in retinal rod outer segments. 1. Biol. Chem. 265. 18867-70. Boesze-Battaglia. K., Hennessey, T. and Albert, A. D. (1989). Cholesterol heterogeneity in bovine rod outer segment disk membranes. 1. Biol. Chem. 264. 8151-S. Fliesler. S. J. and Anderson, A. E. (1983). Chemistry and metabolism of lipids in the verbrate retina. Prog. Lipid Res. 22. 79-131. Folch,J., LeesM. andSloane-Stanley.G. A. (1957). A simple methodfor the isolationand purification of total lipids. I. Biol. Chem.226, 497-509. Hax, W. M. and Van Kessel, W. S.M. ( 1977). Highperformance liquid chromatographic separation and photometric detection of phospholipids.J. Chromatography 142, 73541. House,K.. Badgett,D. and Albert, A. D. (1989). Cholesterol movement between bovine rod outer segmentdisk membranesandphospholipidvesicles.Exp. EyeRes.49. 561-72. Kates,M. (1975). Techniques oj Lipidology.AmericanElsevier Publishing:New York, NY. Litman. B. J. (1973). Lipid model membrane characterization of mixed phospholipid vesicles. Biochemistry 13. 2545-54. Molday, R. and Molday, L. (1987). Differencesin the protein compositionof bovine retinal rod outer segmentdisk andplasmamembraneisolatedby a ricin-gold-dextran density perturbation method. J. Cell Biol. 105, 2589-601. Steck, T., Kezdy, F. and Lange, Y. (1988). An activationco\lision mechanismfor cholesteroltransfer between membranes.1. Biol. Chem.263, 13023-31. Yeagle.P. L. and Young, J. (1986). Factorscontributing to the distribution of cholesterol among phospholipid vesicles.J. Biol. Chem.261, 8175-81. Young. R. W. (1976). Visual cells and the concept of renewal. J. Cdl. Biol. 39, 169-84.
and accepted
in revised
form
27 January
7992)
