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Interactions of Rab5 with Cytosolic Proteins*

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Interactions of Rab5 with Cytosolic Proteins* THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 26, Issue of September 15, pp. 16419-18423,1992 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A. Interactions of rab5 with Cytosolic Proteins* (Received for publication, February 5, 1992) Teymuras V. KurzchaliaS, Jean-Pierre Gorvel, Paul Dupree, RobertParton, Roland Kellner, Tony Houthaeve,Jean Gruenberg, and Kai Simons# From the European Molecular Biology Laboratory, Postfach 10.2209, W-6900Heidelberg, Germany Rab proteins, oneof the subfamilies ofras-like small specificity of the process. Each member of the rab family GTP-binding proteins, are attached to cellular com- investigated so far has been localized to distinct compart- partments or transportvesicles and maydetermine the ments within the cell (Chavrier et al., 1990a; Fischer von specificity of fusion between these compartments and Mollard et al., 1990; Goud et al., 1990; van der Sluijs et al., vesicles. It has been proposed that they alternate be- 1991). Furthermore, rab5 and rabl have been demonstrated tween a membrane-bound and acytosolic state during to control specific transport events in vitro (Gorvel et al., their functional cycle. We have used a photo-cross- 1991; Plutner et al., 1991). linking approach toidentify their cytosolic interaction It has been proposed that the rabproteins alternate between partners. vitro synthesized rab5 was cross-linked In a membrane-boundand a cytosolic location during their func- in the presence of ATP mainly to three cytosolic pro- teins of 52, 65, and 85 kDa. Sucrose density gradient tional cycle (Walworth et al., 1989; Fischer von Mollard et al., centrifugation of the cross-linked products suggested 1991). In this way, the rab may reassociate with the donor that they were part of a 10-14 S complex. Further- compartment via the cytosol after the vesicle has fused with more, rab5 was cross-linked to these and additional the acceptor-compartment membrane. SEC4p, a yeast hom- cytosolic proteins of 42,48, and 160 kDa in the absence ologue of rab proteins, was found plasma membrane-bound, of ATP. Unexpectedly, upon ATP depletionof the cy- vesicular membrane-bound, and in a cytosolic state (Wal- tosol cross-linked and noncross-linked rab5was found worth et al., 1989). Furthermore, there is evidence that cycling in asedimentable high molecular weight structure. of rab3a between a membrane-bound and cytosolic state is Other members of the rab subfamily, but not N-ras, important for the delivery of small synaptic vesicles to the also sedimented under these conditions. Electropho- nerve terminal (Fischer von Mollard et al., 1991). Although it retic and electron microscopic analysis of the pelleted has been shown thatthe hypervariable carboxyl-terminal material revealed that it contained actin filament bun- region of rab proteins plays a role in targeting the proteins to dles and intermediate filaments.Our data suggest that their specific organelles (Chavrier et al., 1991), the molecular cytosolic rab proteins interactwith several proteins in mechanisms of membrane attachment and cycling through a 10-14 S complex, and thatthe rab proteins may interact directly or indirectly via this complex with the cytosol have not been established. the cytoskeleton. Three classes of proteins involved in the functional cycle of the small GTP-binding proteins (ras, rab, and rho) have been identified to date. Theseare the GTPase-activating proteins (GAP),’ GDP dissociation inhibitory protein (GDI), Intracellular transport of proteins in eukaryotic cells is and GDP dissociation stimulating protein (GDS, also called mediated by vesicular carriers (for review, see Pfeffer and GTP exchange protein). There areprobably a family of these Rothman (1987)). These vesicles bud from a donor and spe- proteins, each specific for one or a few closely related small cifically fuse with an acceptor compartment. An important GTP-binding proteins. ras GAPS have been isolated from finding during the last few years was that many different mammals, yeast, and Drosophila. A rho-specific GAP (Morii steps of intracellular transport are GTP-dependent(Salminen et al., 1991) andYptl GAP have been partially purified and Novick, 1987; Melancon et al., 1987; Baker et al., 1988; (Becker et al., 1991), but no rab GAP has yet been reported. Goda and Pfeffer, 1988). According to a model proposed by A GDI has been characterized that acts on rab3a (Matsui et Bourne (1988), each transport step requires a GTP-binding al., 1990) and another that is rho-specific (Fukumoto et al., protein to ensure that a vesicle fuses specifically with its 1990). GDS proteins that act on ras (Wolfman and Macara, target membrane. The recent identification in mammalian 1990) and rab3a (Yamomoto et al., 1990) have also been cells of a large number of small GTP-binding proteins is isolated. The rab proteins as well as ras proteins undergo consistent with this model (Touchot et al., 1987; Haubruck et complex post-translational modifications (Glomset et al., al., 1987; Zahraoui et al., 1989; Chavrier et al., 1990a, 1990b; 1990; Kawata et al., 1990). The modification of ras includes Sewell andKahn, 1988). Among these, therab proteins, isoprenylation of a carboxyl-terminal cysteine residue, pro- belonging to the rassuperfamily, are essential componentsof teolytic removal of carboxyl-terminal amino acids and car- the machinery of vesicular transport and may ensure the boxyl-terminal methylation (Hancock et al., 1989). The en- zymes catalyzing these stepsof rab protein modification have * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby The abbreviations used are: GAP, GTPase activating protein; marked “aduertisement” in accordance with 18 U.S.C. Section 1734 GDI, GDP dissociation inhibitory protein; GDS, GDP dissociation solely to indicate this fact. stimulating protein; AMPPNP, 5’-adenylyl-@,y-imidodiphosphate; $ Current address: Max-Delbriick Centre for Molecular Medicine, GTPyS, guanosine 5’-0-(3-thiotriphosphate;TDBA, 4-(3-trifluoro- Robert-Rossle-Str. 10, W-1115 Berlin-0, Germany. methy1diazirino)benzoic acid Hepes, 4-(2-hydroxyethyl)-l-pipera- 5 To whom correspondenceshould be addressed. Tel.: zineethanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, poly- 49 6221 387334; Fax: 49 6221 387306. acrylamide gel electrophoresis; EF-la, elongation factor-la. 18419 18420 Binding of rab5 to Cytosolic Proteins not yet been well characterized. with silver (Heukeshoven and Dernick, 1988). Preparative two-di- The initial aim of the work described in this paper was to mensional gels of pellets produced from the treatment of 0.5 ml of investigate interaction of rabs with cytosolic proteins. Using cytosol with apyrase were used for microsequence analysis. Amino Acid Sequence Analysis-Protein bands or spots from one- a photocross-linking approach we show that rab5 interacts or two-dimensional gels, respectively, were excised after Coomassie with a number of cytosolic proteins that are in a 10-14 S Blue staining. After washing with water, the gel pieces were lyophi- complex. When ATP was depleted from the cytosol, polym- lized and rehydrated in 100 rl of 100 mM NH4C03, 0.5 mM CaCl' erization of actin and intermediate filaments occurred and containing 1 pg trypsin. After digestion (37 "C for 24 h) peptide the filaments could be recovered in a pellet with rab5 after fragments were extracted from the gel slice with 2 X 100 pl of 1% sucrose gradient centrifugation. This unexpected observation trifluoracetic acid, 0.01% Tween 20, then 70% trifluoroacetic acid, 0.01% Tween 20, and 50% trifluoroacetic acid, acetonitrile, 0.01% may be important in theclarification of functional cycling of Tween 20 (Eckerskorn and Lottspeich, 1989). The combined fractions rabs between membrane and cytosol. were concentrated and subjected to reverse phase high pressure liquid chromatography. EXPERIMENTALPROCEDURES Automated Edman degradation of peptides was performed using Materials-Creatine phosphate, creatine kinase, hexokinase, tryp- an Applied Biosystems sequencer (model 477A) connected to an on- sin, ATP, AMPPNP and GTPyS were purchased from Boehringer line phenyl thiohydantoin analyzer (Applied Biosystems, model 120). Mannheim GmbH (Mannheim, Germany). 4-(3-Trifluoromethyldi- GTP Ouerlay Technique-Pellets from sucrose gradient centrifu- azirino)benzoic acid (TDBA) was a gift from Dr. J. Brunner, Swiss gation were separated by SDS-PAGE and transferred to nitrocellulose Federal Institute of Technology, Zurich, Switzerland. Nt-4-(3-trifluo- using a wet blotting chamber apparatus. The protocol for the GTP romethy1diazirino)benzoyl-Lys (TDBA-Lys)-tRNA was prepared as overlay was essentially that of Lapetina and Reep (1987) but modified described (Wiedmann et al., 1987). The cytosol fraction from baby in our laboratory.' hamster kidney or Madin-Darby canine kidney cells was prepared according a protocol that includes a 200,000 X g centrifugation step RESULTS to remove membranes and polymerized cytoskeletal elements (Gorvel Cross-linking of rub5 to Cytosolic Proteins-We chose rab5 et al., 1991). Cross-linking of in Vitro Synthesized rab Protein-All procedures for our initial investigations. It is localized in the early endo- preceding the UV irradiation step were carried out in dim light. A somes and the plasma membrane and controls early endoso- full-length rab5 cDNA in pGEMl under the T7promoter (Chavrier mal fusion in vitro (Chavrier et al., 1990a; Gorvelet aL, 1991). et
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