December 1992
Volume 314, number 3, 348-350 FEBS 1I895 0 1992 Fcdcration of Eurowan Biochemical Societies 00145793/92/$5.00
Preferential
binding of a-actinin
to actin bundles
Enrico Grazi, Paola Cum-o, Ermes Magi and Christine !kti~ie~bacher Isriruro di CAMea Bioiogicn, Vniversitri di Fcrrara, Via Bursar+48, 44100 Fcrrura, Italy Received 24 Scptcmber
1992; revised version
received 4 November
1992
At 37°C. the a-actinin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol GOOO, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (K,i,I. = Il.3 PM), a-Actinin increases the rigidity of the networks formed by actin bundles in polyethylcnc glycol and by paracrystnlline actin in 16 mM MgCI? but not by F-actin. It is proposed that in the cell a-actinin functions are mostly carried on by interaction with actin bundles. a-Actinin function: Preferential binding; Actin bundle
1. INTRODUCTlON a-Actinin is a 200 kDa protein that crosslinks actin filaments and increases the rigidity of the actin gel. Both phenomena are quite complex and are not completely understood. The binding isotherm of a-actinin with a-ctin, as a function of actin concentration, is anomalous. The apparent association constant decreases with an increase in actin concentration [ 1,2]. This phenomenon was not recognized previously because the effect of actin concentration either was not tested [3-71 or was tested at relatively high nctin concentrations [S]. The critical gelling concentration of a-actinin increases substantially with temperature [3], a phenomenon that led to questioning of the gelling activity of this protein in vivo [S]. it was found, however, that a-actinin is an efficient actin gelling protein, even at 37OC, provided that either the concentration of actin is low (1.22.4 PM) [2] or the reaction mixture is supplemented with macromolecules at a concentration equivalent to that found in the cell sap [9]. We offer evidence here that the presence of a network of actin bundles, independent of the mechanism of its formation, is a prerequisite for a-actinin functioning in vivo. 2. MATERIALS AND METHODS G-actin from rabbit muscle was prepared according to Spudich and Watt [IO] and further gel filtered through Scphadex G-150 [II]. aActinin from chicken gizzard was prepared according to Feramisco and Burridge [l2]. The absorption coefficients used were A:% - 6.2 [ 131 for actin and t1:: = 9.7 [I41 for a-actinin. Molar concentrations were Corresponckwcrnddrew E. Gr.ui,
Istiluto di Chimica Uiologica, Universith di Ferrara. Via Borsari 4G, 44100 Fcrrara, Italy. Fax: (39) (532) 202 723.
348
calculated on the basis of n molecular mass of 42 kDa for octin [IS] and of 200 kDa for 8-actinin 1141.Centrifugation was performed at 37°C in a TLIOO rotor of the TLIOO Beckman centrifuge. Protein was detcrminbd by the Coomassic blue method [IG] as modified by Stoscheck [17]. [“M]N-Ethylmaleimide&tbellcd o-actinin was prepared and rddioactivity determined as previously described [2]. The rigidity of the g:!s of actin was mcasurcd by the droplets method [IS].
3. RESULTS 3.1. Effeect
of pol~wthyle~~e
interactions
&cd 6000 on the camplex of a-actthin with actirl
It is known that, in the presence of polyethylene glycol (PEG) 6000, F-actin undergoes massive conversion into actin bundles [19]. At 12 ,uM actin the boundary between filaments and bundles ranges between 6 and 7% (w/v) PEG. Addition of 0.2 PM a-actinin to the system displaces the boundary toward a lower (4-G%) PEG concentration. This shows that a-actinin favours actin bundling (Fig. 1). The amount of a-actinin co-sedimenting with F-actin is not influenced up to 3% PEG but increases at larger PEG concentrations. The increase is concomitant with the formation of actin bundles. Under these conditions (6.7% PEG), approximately the same amount of a-actinin is sedimented by centrifugation either at 9,900 x g (actin bundles are collected) or at 36&000 xg (actin bundles plus actin filaments are collected). Thus, at this PEG concentration, a-actinin is bound almost exclusively to actin bundles (Fig. 2). The a-actinin-F-actin binding isotherm is anomalous both in the absence [1,2] and in the presence of 3% PEG (Fig. 3). Under both these conditions actin is filamentous. The binding isotherm becomes hyperbolic concomitant with the formation of actin bundles (6.7% PEG). Double reciprocal plot analysis shows that a sin-
Volume 314, number
FEBS LETTERS
3
December 1992
IACTIAI(,ufV A
rl
A
AA Ls IPOLYETHYLENE GLYCOLl ct,w/v )
Fig. I, In polyethylene glycol GO00a-actinin promotes actin bundling. The mixtures contained F-actin (12 ,uM as monomer), 0.5 mM ATP. 2 nM Mf$&, 0.1 M KCI, 1 mM dithiothrcitol, 10 mM Tris-HCI. PEG GO00(w/v) as indicated in thr: figure, with (0) or without (0) 0.2 PM [‘H]N-ethylmalcimidc-lab&d a-actinin (spechic activity 12,000 dpml nmol), pH 7.5. Aflcr 60 min of incubation at 37’C the mixtures were centrift@ for 10 min at 9,900 x g to sediment actin bundles, and the supcrnatant solutions were as& for protein.
gle dissociation constant (KdiSS, = 11.3 ,KM) accounts for the binding of 0.185 ,uM out of the total 0.2 ,uM aactinin (Fig. 3b).
0
O
/._
0.1
0.2
0.3
0.11
lo-’ x l/[ACTlNl(R-‘1 PiB. 3. Binding of or-nctinin to F-actin in polyclhylcne dycol GPO. as a function of actin concentration. (a) Actin concentration was as indicated in the figure; PEG was cithcr 3% (A) or 6.7% (0.0). O!her conditions were as described in Fig. I. After 60 min of incubation at 37’C, the mixtures were ccntrifuugd for 10 min cithcr at 9,900 x g (@j or at 3GG,OOO x g (0. A). The pellets were then assayed for labcllU.i a-actinin. (b) Double reciprocal plot of the binding of a-actinin lo F-actiu.
3.2. Tlw iweractiorz of a-actinin with parucrystalline
aeth in I6 mM MgCi2 the presence of 0.2 yM a-actinin (total concentration) and of 48 ,uM actin, more c+actinin is bound to paracrystalline actin in 16 mM MgCIz (0.091 yM, Fig. 4) than to filamentous actin in 2 mM MgQ plus 0.1 M KC1 (0.026 ,uM, Fig. 3). The binding of a-actinin to In
pararrystailine actin, however, is not described by a simple hyperbolic function (Fig. 4). The rigidity of the system (7.1 ,uM actin as monomer) increases from 2.6 to 6.7 dynkm’ in the transition from
h A4 A
A
A
1
0
,I
0
lo+
I
2
0.1
i
6
II'OLYETIIYLENE GLYCflLItZ.n/u)
Fig. 2. Binding of or-actinin to F-nctin in polyethylene glycol 6000. Eapcrimentnl conditions were as described in Fig. I, AfIcr 60 min of incubation at 37OC. the mixtures were centrifuged for 10 min either at 9,900 x g (A) or at 35G,OOO x g (A). The pcllcts were then assayed for labelled a-actinin.
0.2 x 1/[ACTINI
0.3
0.4
(N-1)
FiB. 4. Double reciprocal plot of the binding of a-actinin to actin paracrystals in 16 mM M@&. The mixtures conuincd F-actin as indicated in the figure,0.2 PM ~“H]N-ethylmaleimidc-labcllcd a-actinin (spccilic activity 12,000 dpminmoij, 0.5 mivi ATT. i6 mM M@&, I mM dithiothrcitol, 10 mM Tris-HCI, pH 7.5. After 60 min of incubation at 37°C. the mixtures wcrc ccntrifu&?d for 10 min al 366,000 x g. The pellets were then assayed for labclled a-actinin.
349
FIBS LETTERS
Volume 314, number 3
ii 3
0
10
20
30
40
50
the system is increased by cc-actinin, even at 37°C. In F-actin, at least at 37’C, the monodentate binding of cc-actinin prevails. This is indicated by the total lack of effect of a-actinin on the rigidity of the network formed by F-actin. These observations support the view that, in the cell, a-actinin functions are mostly carried on by interaction with actin bundles.
z
0
1
December I992
lo
n~k/rol~,lrrlSrrrlcnls: This work was supported by grants of MURST 40% and 60%.
TOTAL d-ACTININ
(nil) Fig. 5. Estimate of the effect of increasiug a-actinin concentrations on the rigidity of the network formed by paracrystalline actin. The mixtures contained F-nctin (7.1 PM as monomer), 0.5 rnM ATP, I mM dithiothrcitol. IO mM Tris-HCI and either 16 mM M&l2 (O or 2 mM MgCIz plus 0.1 M KC1 (0). a-Actinin concentration was as indicated in the figure. After GOmin of incubation at 37°C and pH 7.5, rigidity measurements were performed. d density rcprcscnts the difference between the density at which the droplets remained stationary in the complete system and in the salt solution without protein.
to paracrystalline actin. The rigidity is further increased to 24.3 dyn/cm? when paracrystalline actin is supplemented with 0.05 PM a-actinin (Fig. 5). F-actin
4. DISCUSSION In 6.7% PEG 6000, the binding of a-actinin to actin bundles is described by a single dissociation constant of I 1.3 PM. This contrasts with the anomalous behaviour displayed by F-actin and characterized by the apparent decrease of the binding constant to cr.-actinin, as a function of the increase of F-actin concentration. The parallel arrays of actin filaments, formed either in 6.7% PEG GO00or in 16 mM MgCI,, bind a-actinin tighter than does F-actin. This is in keeping with the observation that, in the cell, a-actinin is mostly associated with actin fibers [20,213. It is likely that the arrays of filaments offer an ordered matrix of actin, which favours by bidentate binding of a-actinin. The crosslinking by #-actinin prevents the filaments from sliding in actin bundles. As a result, since the network of actin bundles is largely anastomosed, the rigidity of
350
REFERENCES [II Grazi, E., Trombeua, G. and Ciuidoboni, M. (1990) Biochem, Int. 21, 633-640.
[?I Grazi, E., Trombetta, G. and Guidoboni, M. (1991) J. Muscle
Rcs. Cell Motil. 12, 579-584. [31 Gall, D.E., Sun&i, A., Temple. J. &nd llolmes, G.R. (1972) J. Mol. Biol. G7, 469-488. 141Jockusch, B.M. and Isenbcrg, G. (1981) Proc. N31. Acad. Sci. USA 78,3005-3009. 151 Bennett, J.P., Scott-Zaner, K..&~:-Stoss& T.P. (19S4) Biochcmistry 23, 5081-508G. PI Ohtaki, T., Tsukita, S., Mimura, N.. Tsukita, S. and Asano, A. (1955) Eur. J. Biochem. 153, 609-620. I71 Landon, F., Gache, Y., Touitou, H. and Olomucki, A. (19S5) Eur. J. Biochcm. 153, 231-237. iSI Sate, M. Schwurz, W.H. and Pollard, T.D. (1987) Nature 335, 825-830, PI Grazi. E.. Trombctta, G.. Magri, E. and Cuneo, P. (1990) FEBS Lelt. 272, 149-151. WI Spudich, J.A. and Watt, S. (1971) J. Biol. Chcm. 246,486&4871. 1111McLean-Flcchter, S. and Pollard, T.D. (1980) Biocbcm. Biophys. Res. Commun. 9G, 18-27. 1121Feramisco, J.R. and Burridge, K. (1980) J. Biol. Chem. 255, 1194-l 199. 1131 Gordon, D.J., Yang, Y.Z. and Korn, E.D. (1976) 1. Biol. Chem. 25 I, 7474-7479. 1141 Suzuki, A., Gall. D.E., Singh, 1.. Allen. R.E., Robson, R.M. and Stromcr, M.H. (1976) J. Biol. Chcm. 251, G8GO-6870. 1151 Collins, J.H. and Elzingaa, M. (1975) I. Biol. Chem. 250, 59155920. Bradford, M.M. (1976) Anal. Biochcm. 72, 348-254. ;;:; Stoschcck. CM. (1990) Anal. Biochem. 184, 11l-l IG. [IW Grazi, E., Magi. E., Cuneo. P. and Catnldi, A. (1991) FEBS Lett. 295, 163-lGG. u91 Suzuki, A., Yamazaki, M. and Ito, T. (1989) Uiochemistry 38, G513-6518. Lazuridcs, E. (1976) J. Cell Biol. 68, 202-219. ;;:; Brctschcr. A. and Weher, K. [19+/S)1. Cell Biol. 79, 339-345.
Volume 314, number 3, 348-350 FEBS 1I895 0 1992 Fcdcration of Eurowan Biochemical Societies 00145793/92/$5.00
Preferential
binding of a-actinin
to actin bundles
Enrico Grazi, Paola Cum-o, Ermes Magi and Christine !kti~ie~bacher Isriruro di CAMea Bioiogicn, Vniversitri di Fcrrara, Via Bursar+48, 44100 Fcrrura, Italy Received 24 Scptcmber
1992; revised version
received 4 November
1992
At 37°C. the a-actinin-F-actin binding isotherm is anomalous. In 6.7% polyethylene glycol GOOO, concomitantly with the formation of actin bundles, the binding isotherm becomes hyperbolic (K,i,I. = Il.3 PM), a-Actinin increases the rigidity of the networks formed by actin bundles in polyethylcnc glycol and by paracrystnlline actin in 16 mM MgCI? but not by F-actin. It is proposed that in the cell a-actinin functions are mostly carried on by interaction with actin bundles. a-Actinin function: Preferential binding; Actin bundle
1. INTRODUCTlON a-Actinin is a 200 kDa protein that crosslinks actin filaments and increases the rigidity of the actin gel. Both phenomena are quite complex and are not completely understood. The binding isotherm of a-actinin with a-ctin, as a function of actin concentration, is anomalous. The apparent association constant decreases with an increase in actin concentration [ 1,2]. This phenomenon was not recognized previously because the effect of actin concentration either was not tested [3-71 or was tested at relatively high nctin concentrations [S]. The critical gelling concentration of a-actinin increases substantially with temperature [3], a phenomenon that led to questioning of the gelling activity of this protein in vivo [S]. it was found, however, that a-actinin is an efficient actin gelling protein, even at 37OC, provided that either the concentration of actin is low (1.22.4 PM) [2] or the reaction mixture is supplemented with macromolecules at a concentration equivalent to that found in the cell sap [9]. We offer evidence here that the presence of a network of actin bundles, independent of the mechanism of its formation, is a prerequisite for a-actinin functioning in vivo. 2. MATERIALS AND METHODS G-actin from rabbit muscle was prepared according to Spudich and Watt [IO] and further gel filtered through Scphadex G-150 [II]. aActinin from chicken gizzard was prepared according to Feramisco and Burridge [l2]. The absorption coefficients used were A:% - 6.2 [ 131 for actin and t1:: = 9.7 [I41 for a-actinin. Molar concentrations were Corresponckwcrnddrew E. Gr.ui,
Istiluto di Chimica Uiologica, Universith di Ferrara. Via Borsari 4G, 44100 Fcrrara, Italy. Fax: (39) (532) 202 723.
348
calculated on the basis of n molecular mass of 42 kDa for octin [IS] and of 200 kDa for 8-actinin 1141.Centrifugation was performed at 37°C in a TLIOO rotor of the TLIOO Beckman centrifuge. Protein was detcrminbd by the Coomassic blue method [IG] as modified by Stoscheck [17]. [“M]N-Ethylmaleimide&tbellcd o-actinin was prepared and rddioactivity determined as previously described [2]. The rigidity of the g:!s of actin was mcasurcd by the droplets method [IS].
3. RESULTS 3.1. Effeect
of pol~wthyle~~e
interactions
&cd 6000 on the camplex of a-actthin with actirl
It is known that, in the presence of polyethylene glycol (PEG) 6000, F-actin undergoes massive conversion into actin bundles [19]. At 12 ,uM actin the boundary between filaments and bundles ranges between 6 and 7% (w/v) PEG. Addition of 0.2 PM a-actinin to the system displaces the boundary toward a lower (4-G%) PEG concentration. This shows that a-actinin favours actin bundling (Fig. 1). The amount of a-actinin co-sedimenting with F-actin is not influenced up to 3% PEG but increases at larger PEG concentrations. The increase is concomitant with the formation of actin bundles. Under these conditions (6.7% PEG), approximately the same amount of a-actinin is sedimented by centrifugation either at 9,900 x g (actin bundles are collected) or at 36&000 xg (actin bundles plus actin filaments are collected). Thus, at this PEG concentration, a-actinin is bound almost exclusively to actin bundles (Fig. 2). The a-actinin-F-actin binding isotherm is anomalous both in the absence [1,2] and in the presence of 3% PEG (Fig. 3). Under both these conditions actin is filamentous. The binding isotherm becomes hyperbolic concomitant with the formation of actin bundles (6.7% PEG). Double reciprocal plot analysis shows that a sin-
Volume 314, number
FEBS LETTERS
3
December 1992
IACTIAI(,ufV A
rl
A
AA Ls IPOLYETHYLENE GLYCOLl ct,w/v )
Fig. I, In polyethylene glycol GO00a-actinin promotes actin bundling. The mixtures contained F-actin (12 ,uM as monomer), 0.5 mM ATP. 2 nM Mf$&, 0.1 M KCI, 1 mM dithiothrcitol, 10 mM Tris-HCI. PEG GO00(w/v) as indicated in thr: figure, with (0) or without (0) 0.2 PM [‘H]N-ethylmalcimidc-lab&d a-actinin (spechic activity 12,000 dpml nmol), pH 7.5. Aflcr 60 min of incubation at 37’C the mixtures were centrift@ for 10 min at 9,900 x g to sediment actin bundles, and the supcrnatant solutions were as& for protein.
gle dissociation constant (KdiSS, = 11.3 ,KM) accounts for the binding of 0.185 ,uM out of the total 0.2 ,uM aactinin (Fig. 3b).
0
O
/._
0.1
0.2
0.3
0.11
lo-’ x l/[ACTlNl(R-‘1 PiB. 3. Binding of or-nctinin to F-actin in polyclhylcne dycol GPO. as a function of actin concentration. (a) Actin concentration was as indicated in the figure; PEG was cithcr 3% (A) or 6.7% (0.0). O!her conditions were as described in Fig. I. After 60 min of incubation at 37’C, the mixtures were ccntrifuugd for 10 min cithcr at 9,900 x g (@j or at 3GG,OOO x g (0. A). The pellets were then assayed for labcllU.i a-actinin. (b) Double reciprocal plot of the binding of a-actinin lo F-actiu.
3.2. Tlw iweractiorz of a-actinin with parucrystalline
aeth in I6 mM MgCi2 the presence of 0.2 yM a-actinin (total concentration) and of 48 ,uM actin, more c+actinin is bound to paracrystalline actin in 16 mM MgCIz (0.091 yM, Fig. 4) than to filamentous actin in 2 mM MgQ plus 0.1 M KC1 (0.026 ,uM, Fig. 3). The binding of a-actinin to In
pararrystailine actin, however, is not described by a simple hyperbolic function (Fig. 4). The rigidity of the system (7.1 ,uM actin as monomer) increases from 2.6 to 6.7 dynkm’ in the transition from
h A4 A
A
A
1
0
,I
0
lo+
I
2
0.1
i
6
II'OLYETIIYLENE GLYCflLItZ.n/u)
Fig. 2. Binding of or-actinin to F-nctin in polyethylene glycol 6000. Eapcrimentnl conditions were as described in Fig. I, AfIcr 60 min of incubation at 37OC. the mixtures were centrifuged for 10 min either at 9,900 x g (A) or at 35G,OOO x g (A). The pcllcts were then assayed for labelled a-actinin.
0.2 x 1/[ACTINI
0.3
0.4
(N-1)
FiB. 4. Double reciprocal plot of the binding of a-actinin to actin paracrystals in 16 mM M@&. The mixtures conuincd F-actin as indicated in the figure,0.2 PM ~“H]N-ethylmaleimidc-labcllcd a-actinin (spccilic activity 12,000 dpminmoij, 0.5 mivi ATT. i6 mM M@&, I mM dithiothrcitol, 10 mM Tris-HCI, pH 7.5. After 60 min of incubation at 37°C. the mixtures wcrc ccntrifu&?d for 10 min al 366,000 x g. The pellets were then assayed for labclled a-actinin.
349
FIBS LETTERS
Volume 314, number 3
ii 3
0
10
20
30
40
50
the system is increased by cc-actinin, even at 37°C. In F-actin, at least at 37’C, the monodentate binding of cc-actinin prevails. This is indicated by the total lack of effect of a-actinin on the rigidity of the network formed by F-actin. These observations support the view that, in the cell, a-actinin functions are mostly carried on by interaction with actin bundles.
z
0
1
December I992
lo
n~k/rol~,lrrlSrrrlcnls: This work was supported by grants of MURST 40% and 60%.
TOTAL d-ACTININ
(nil) Fig. 5. Estimate of the effect of increasiug a-actinin concentrations on the rigidity of the network formed by paracrystalline actin. The mixtures contained F-nctin (7.1 PM as monomer), 0.5 rnM ATP, I mM dithiothrcitol. IO mM Tris-HCI and either 16 mM M&l2 (O or 2 mM MgCIz plus 0.1 M KC1 (0). a-Actinin concentration was as indicated in the figure. After GOmin of incubation at 37°C and pH 7.5, rigidity measurements were performed. d density rcprcscnts the difference between the density at which the droplets remained stationary in the complete system and in the salt solution without protein.
to paracrystalline actin. The rigidity is further increased to 24.3 dyn/cm? when paracrystalline actin is supplemented with 0.05 PM a-actinin (Fig. 5). F-actin
4. DISCUSSION In 6.7% PEG 6000, the binding of a-actinin to actin bundles is described by a single dissociation constant of I 1.3 PM. This contrasts with the anomalous behaviour displayed by F-actin and characterized by the apparent decrease of the binding constant to cr.-actinin, as a function of the increase of F-actin concentration. The parallel arrays of actin filaments, formed either in 6.7% PEG GO00or in 16 mM MgCI,, bind a-actinin tighter than does F-actin. This is in keeping with the observation that, in the cell, a-actinin is mostly associated with actin fibers [20,213. It is likely that the arrays of filaments offer an ordered matrix of actin, which favours by bidentate binding of a-actinin. The crosslinking by #-actinin prevents the filaments from sliding in actin bundles. As a result, since the network of actin bundles is largely anastomosed, the rigidity of
350
REFERENCES [II Grazi, E., Trombeua, G. and Ciuidoboni, M. (1990) Biochem, Int. 21, 633-640.
[?I Grazi, E., Trombetta, G. and Guidoboni, M. (1991) J. Muscle
Rcs. Cell Motil. 12, 579-584. [31 Gall, D.E., Sun&i, A., Temple. J. &nd llolmes, G.R. (1972) J. Mol. Biol. G7, 469-488. 141Jockusch, B.M. and Isenbcrg, G. (1981) Proc. N31. Acad. Sci. USA 78,3005-3009. 151 Bennett, J.P., Scott-Zaner, K..&~:-Stoss& T.P. (19S4) Biochcmistry 23, 5081-508G. PI Ohtaki, T., Tsukita, S., Mimura, N.. Tsukita, S. and Asano, A. (1955) Eur. J. Biochem. 153, 609-620. I71 Landon, F., Gache, Y., Touitou, H. and Olomucki, A. (19S5) Eur. J. Biochcm. 153, 231-237. iSI Sate, M. Schwurz, W.H. and Pollard, T.D. (1987) Nature 335, 825-830, PI Grazi. E.. Trombctta, G.. Magri, E. and Cuneo, P. (1990) FEBS Lelt. 272, 149-151. WI Spudich, J.A. and Watt, S. (1971) J. Biol. Chcm. 246,486&4871. 1111McLean-Flcchter, S. and Pollard, T.D. (1980) Biocbcm. Biophys. Res. Commun. 9G, 18-27. 1121Feramisco, J.R. and Burridge, K. (1980) J. Biol. Chem. 255, 1194-l 199. 1131 Gordon, D.J., Yang, Y.Z. and Korn, E.D. (1976) 1. Biol. Chem. 25 I, 7474-7479. 1141 Suzuki, A., Gall. D.E., Singh, 1.. Allen. R.E., Robson, R.M. and Stromcr, M.H. (1976) J. Biol. Chcm. 251, G8GO-6870. 1151 Collins, J.H. and Elzingaa, M. (1975) I. Biol. Chem. 250, 59155920. Bradford, M.M. (1976) Anal. Biochcm. 72, 348-254. ;;:; Stoschcck. CM. (1990) Anal. Biochem. 184, 11l-l IG. [IW Grazi, E., Magi. E., Cuneo. P. and Catnldi, A. (1991) FEBS Lett. 295, 163-lGG. u91 Suzuki, A., Yamazaki, M. and Ito, T. (1989) Uiochemistry 38, G513-6518. Lazuridcs, E. (1976) J. Cell Biol. 68, 202-219. ;;:; Brctschcr. A. and Weher, K. [19+/S)1. Cell Biol. 79, 339-345.
