rab4 Function in Membrane Recycling from Early Endosomes Depends on a Membrane to Cytoplasm Cycle
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m203064200
ISSN1083-351X
AutoresKarin Mohrmann, Lisya Gerez, Viola Oorschot, Judith Klumperman, Peter van der Sluijs,
Tópico(s)Lysosomal Storage Disorders Research
ResumoThe monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase. The monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase. soluble NSF attachment protein receptor vesicle-soluble NSF attachment protein receptor X31 influenza hemagglutinin Madin-Darby canine kidney Chinese hamster ovary transferrin Myc-tagged human transferrin receptor post nuclear supernatant cellubrevin methionine/cysteine-free medium phosphate-free medium GDP dissociation inhibitor modified Eagle's medium phosphate-buffered saline 4-morpholineethanesulfonic acid endoplasmic reticulum Small GTPases of the rab family are key regulators of vesicular transport in eukaryotic cells. More than 60 rab proteins have been identified, many of which are localized to intracellular organelles of the central vacuolar system (see Refs. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar and 2Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (369) Google Scholar). The active GTP-bound form of rab proteins associates with multiple other proteins, which may serve to relay the GTPase switch to effector systems such as SNARE1 complexes involved in membrane docking and fusion (see Refs. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar and 3Chen Y.A. Scheller R.H. Nature Rev. Mol. Cell. Biol. 2001; 2: 98-106Crossref PubMed Scopus (853) Google Scholar) and the cytoskeleton for the coordination of organelle motility (4Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (409) Google Scholar, 5Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 1: 376-382Crossref PubMed Scopus (393) Google Scholar, 6Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). It is thought that rab proteins may work as timers whose set point is determined by the relative rates of GTP hydrolysis and GDP exchange (7Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M. Goody R. Zerial M. Nature. 1996; 383: 266-269Crossref PubMed Scopus (267) Google Scholar). rab GTPases cycle on and off membranes through the activity of a protein known as GDP dissociation inhibitor (GDI). GDI extracts the GDP form of rab proteins from membrane, shields their lipophilic carboxyl terminus from the aqueous cytoplasm, and can present complexed rab protein again to a donor membrane for a new activity cycle (see Ref. 8Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). The significance of a membrane to cytoplasm cycle for rab activity in the biosynthetic pathway has been called into question recently. Evidence for this notion derives from studies with Ypt1p chimeras in which the hydrophobic tails of the v-SNAREs Sec22p and Snc2p replaced the Ypt1p carboxyl terminus. These chimeric proteins appear to be correctly targeted and retain the function of Ypt1p in the biosynthetic pathway (9Ossig R. Laufer W. Schmitt H.D. Gallwitz D. EMBO J. 1995; 14: 3645-3653Crossref PubMed Scopus (32) Google Scholar), although they are resistant to GDI extraction (10Cao X. Barlowe C. J. Cell Biol. 2000; 149: 55-65Crossref PubMed Scopus (116) Google Scholar).We have been investigating the role of rab4 in transport through the early endocytic pathway. rab4 regulates recycling from early endosomes (11van der Sluijs P. Hull M. Webster P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 12Roberts M. Barry S. Woods A. van der Sluijs P. Norman J. Curr. Biol. 2001; 11: 1392-1402Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar) and is localized to vacuolar and tubular subdomains of early endocytic compartments and transport vesicles but not to the plasma membrane (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 14van der Sluijs P. Hull M. Zahraoui A. Tavitian A. Goud B. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6313-6317Crossref PubMed Scopus (219) Google Scholar, 15de Wit H. Lichtenstein Y. Geuze H. Kelly R.B. van der Sluijs P. Klumperman J. Mol. Biol. Cell. 1999; 10: 4163-4176Crossref PubMed Scopus (53) Google Scholar). rab4 is phosphorylated on Ser-196 during mitosis (16Bailly E. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Crossref PubMed Scopus (130) Google Scholar, 17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). This increases the cytoplasmic pool ∼5–10-fold, possibly due to the interaction with the mitotic peptidyl-prolyl isomerase Pin1 (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Surprisingly, these phosphorylated rab4 molecules are in the GTP form. Because the cytoplasmic pool of rab proteins is thought to be in the GDP-bound form, this suggests that the additional rab4 molecules in the cytoplasm of mitotic cells are dissociated from early endosomes. Perturbation of the normal rab4 cycle by phosphorylation of either the cytoplasmic or membrane-bound pools may result in an increased cytoplasmic steady-state localization during mitosis. Hence, it has been difficult to establish whether membrane-bound rab4 is a target for mitotic kinases. To address this question, and to investigate whether membrane transport through early endosomes depends on cycles of rab4 dissociation and association with early endosomes, we constructed NHrab4cbvn in which we replaced the carboxyl-terminal Cys-Gly-Cys motif of rab4 with the transmembrane domain of cellubrevin, an endosomal v-SNARE. We found that NHrab4cbvn was fixed permanently on endosomal membranes and did not support rab4 function in membrane transport. The chimeric protein was phosphorylated during mitosis, showing that membrane-bound rab4 is targeted by mitotic kinases.DISCUSSIONProtein phosphorylation plays a key role in the regulation of membrane transport and organelle inheritance during the mammalian cell cycle. Intracellular transport is coordinately inhibited in dividing cells, and organelle fragmentation is thought to occur due to ongoing transport vesicle formation while membrane fusion is inhibited. The molecular mechanisms underlying the inhibition of membrane transport during mitosis are only partially understood. rab4 regulates membrane transport through early endosomes and is phosphorylated by p34cdc2 kinase (11van der Sluijs P. Hull M. Webster P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Thus rab4 is likely to be one of the targets for the inhibition of endocytic transport. Indeed, although rab4 is associated with endosomes in interphase, during mitosis it is localized to the cytoplasm in a complex with the peptidyl prolylisomerase Pin1 and presumably unable to perform its endosomal function (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Why phosphorylated rab4 is localized to the cytoplasm is not clear. Phosphorylation of the cytoplasmic rab4 pool might inhibit its recruitment to endosomes. Alternatively, phosphorylation of membrane-bound rab4 may enhance its dissociation into the cytoplasm. Both scenarios or a combination thereof will result in the depletion of rab4 from early endosomes and the build-up of a cytoplasmic pool of rab4 molecules. A rab4 truncation mutant that is constitutively expressed in the cytoplasm does become phosphorylated during mitosis, which is consistent with a model in which phosphorylated cytoplasmic rab4 molecules fail to be recruited to endosomes (17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Informative as it is, this result cannot be used as evidence against the second model, because it is not known whether the endosomal pool of rab4 might be targeted by a mitotic kinase.We addressed this question in expression experiments with an rab4 construct that was permanently attached to early endosomes, because its consensus sequence for carboxyl-terminal isoprenylation was replaced by the transmembrane domain of the endosomal v-SNARE, cellubrevin. The chimeric protein was membrane-associated in both interphase and mitotic cells. During mitosis NHrab4cbvn was phosphorylated, showing for the first time that phosphorylation of rab4 can also occur on membranes. This also explains the previous finding that phosphorylated cytoplasmic rab4 is in the GTP-bound form (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Because rab4 guanine nucleotide exchange activity remains membrane-associated during mitosis (32Ayad N. Hull M. Mellman I. EMBO J. 1997; 16: 4497-4507Crossref PubMed Scopus (48) Google Scholar), enhanced dissociation of phosphorylated rab4-GTP from endosomes increases the number of cytoplasmic rab4 molecules in the GTP-bound form.GTP hydrolysis on rab proteins was initially thought to be required for membrane docking and fusion or recycling of the GDP-bound form back to the donor organelle (39Bourne H.R. Cell. 1988; 53: 669-671Abstract Full Text PDF PubMed Scopus (340) Google Scholar). More recent experiments with Ypt1p and Sec4p that were permanently attached to membranes have called this concept into question (9Ossig R. Laufer W. Schmitt H.D. Gallwitz D. EMBO J. 1995; 14: 3645-3653Crossref PubMed Scopus (32) Google Scholar). These mutants appeared to be targeted to the correct membranes and reportedly retained the capacity to maintain vesicular transport through the biosynthetic pathway. In contrast to these rab proteins that act between the ER and the plasma membrane, our recycling experiments in MDCK cells, however, showed that NHrab4cbvn cannot substitute for wild type rab4.Because rab proteins are important for tethering of transport vesicles to their target membrane (see Ref. 40Brennwald P. J. Cell Biol. 2000; 149: 1-3Crossref PubMed Google Scholar) and for their formation at the donor compartment (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 41Nuoffer C. Davidson H. Matteson J. Meinkoth J. Balch W.E. J. Cell Biol. 1994; 125: 225-238Crossref PubMed Scopus (191) Google Scholar, 42McLaughlan H. Newell J. Morrice N. Osborne A. West M. Smythe E. Curr. Biol. 1998; 8: 34-45Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 43Carroll K.S. Hanna J. Simon I. Krise J. Barbero P. Pfeffer S.R. Science. 2001; 292: 1373-1376Crossref PubMed Scopus (202) Google Scholar), a salvage pathway is required to recycle rab proteins from the acceptor membrane. GDI-mediated extraction followed by cytosolic transfer of a rab·rab GDI complex back to the donor or transport vesicle is an attractive mechanism that is supported by experimental evidence, although it is also possible that retrieval to the donor membrane may occur via a retrograde transport pathway. The observation, that NHrab4cbvn permanently resides on membranes and has an inhibitory phenotype in the transport assay, suggests that a cycle of rab4 between membrane and cytosol is important for its function in membrane recycling from early endosomes. It is unlikely that retrieval of NHrab4cbvn to early endosomes proceeds via the cell surface and endocytosis, because immunoelectron microscopy shows that neither rab4 (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 15de Wit H. Lichtenstein Y. Geuze H. Kelly R.B. van der Sluijs P. Klumperman J. Mol. Biol. Cell. 1999; 10: 4163-4176Crossref PubMed Scopus (53) Google Scholar) nor NHrab4cbvn localize to the plasma membrane.Why would rab4 behave differently in this respect than do Ypt1p and Sec4p? At least two explanations can be put forward to account for this disparity. First, even though the biosynthetic and endocytic pathways are regulated by members of conserved protein families, there appear to be clear differences. For instance, deletion of the YPT1 orSEC4 genes in yeast is lethal (44Segev N. Mullholland J. Botstein D. Cell. 1988; 52: 915-924Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 45Salminen A. Novick P.J. Cell. 1987; 49: 527-538Abstract Full Text PDF PubMed Scopus (589) Google Scholar), whereas knockout strains of YPT5, whose product regulates the endocytic pathway, are viable (46Horazdovski B.F. Busch G.R. Emr S. EMBO J. 1994; 13: 1297-1309Crossref PubMed Scopus (168) Google Scholar). Second, because rab GTPases exert their functions in membrane transport through multiple effector proteins (reviewed in Ref. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar), this may simply indicate that there is not necessarily a common principle by which rab proteins control membrane traffic. An example of this idea is provided by rab27a, which in melanocytes cooperates with myosin Va (6Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar, 47Wu X.S. Rao K. Zhang H. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 1-8Crossref PubMed Scopus (379) Google Scholar) but not in cytotoxic T lymphocytes (48Haddad E.K., Wu, X. Hammer III, J.A. Henkart P.A. J. Cell Biol. 2001; 152: 835-842Crossref PubMed Scopus (208) Google Scholar). Presently, five rab4 effector proteins have been characterized. These are rabaptin-4 (49Nagelkerken B. van Anken E. van Raak M. Gerez L. Mohrmann K. van Uden N. Holthuizen J. Pelkmans L. van der Sluijs P. Biochem. J. 2000; 346: 593-601Crossref PubMed Scopus (55) Google Scholar), rabaptin-5 (50Vitale G. Rybin V. Christoforidis S. Thornqvist P.O. McCaffrey M. Stenmark H. Zerial M. EMBO J. 1998; 17: 1941-1951Crossref PubMed Scopus (195) Google Scholar), rabenosyn-5 (51de Renzis S. Sönnichsen B. Zerial M. Nat. Cell Biol. 2002; 4: 124-133Crossref PubMed Scopus (260) Google Scholar), rabip4 (52Cormont M. Mari M. Galmiche A. Hofman P. Le Marchand-Brustel Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1637-1642Crossref PubMed Scopus (85) Google Scholar), and the cytoplasmic dynein light intermediate chain-1 (53Bielli A. Thörnqvist P.O. Hendrick A.G. Finn R. Fitzgerald K. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2001; 281: 1141-1153Crossref PubMed Scopus (77) Google Scholar), the first three of which are bifunctional ones that also interact with rab5. Clearly a further understanding of membrane recycling via early endosomes requires the identification and characterization of additional rab4 effector and accessory proteins. Small GTPases of the rab family are key regulators of vesicular transport in eukaryotic cells. More than 60 rab proteins have been identified, many of which are localized to intracellular organelles of the central vacuolar system (see Refs. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar and 2Pereira-Leal J.B. Seabra M.C. J. Mol. Biol. 2000; 301: 1077-1087Crossref PubMed Scopus (369) Google Scholar). The active GTP-bound form of rab proteins associates with multiple other proteins, which may serve to relay the GTPase switch to effector systems such as SNARE1 complexes involved in membrane docking and fusion (see Refs. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar and 3Chen Y.A. Scheller R.H. Nature Rev. Mol. Cell. Biol. 2001; 2: 98-106Crossref PubMed Scopus (853) Google Scholar) and the cytoskeleton for the coordination of organelle motility (4Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Science. 1998; 279: 580-585Crossref PubMed Scopus (409) Google Scholar, 5Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 1: 376-382Crossref PubMed Scopus (393) Google Scholar, 6Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). It is thought that rab proteins may work as timers whose set point is determined by the relative rates of GTP hydrolysis and GDP exchange (7Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M. Goody R. Zerial M. Nature. 1996; 383: 266-269Crossref PubMed Scopus (267) Google Scholar). rab GTPases cycle on and off membranes through the activity of a protein known as GDP dissociation inhibitor (GDI). GDI extracts the GDP form of rab proteins from membrane, shields their lipophilic carboxyl terminus from the aqueous cytoplasm, and can present complexed rab protein again to a donor membrane for a new activity cycle (see Ref. 8Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). The significance of a membrane to cytoplasm cycle for rab activity in the biosynthetic pathway has been called into question recently. Evidence for this notion derives from studies with Ypt1p chimeras in which the hydrophobic tails of the v-SNAREs Sec22p and Snc2p replaced the Ypt1p carboxyl terminus. These chimeric proteins appear to be correctly targeted and retain the function of Ypt1p in the biosynthetic pathway (9Ossig R. Laufer W. Schmitt H.D. Gallwitz D. EMBO J. 1995; 14: 3645-3653Crossref PubMed Scopus (32) Google Scholar), although they are resistant to GDI extraction (10Cao X. Barlowe C. J. Cell Biol. 2000; 149: 55-65Crossref PubMed Scopus (116) Google Scholar). We have been investigating the role of rab4 in transport through the early endocytic pathway. rab4 regulates recycling from early endosomes (11van der Sluijs P. Hull M. Webster P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 12Roberts M. Barry S. Woods A. van der Sluijs P. Norman J. Curr. Biol. 2001; 11: 1392-1402Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar) and is localized to vacuolar and tubular subdomains of early endocytic compartments and transport vesicles but not to the plasma membrane (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 14van der Sluijs P. Hull M. Zahraoui A. Tavitian A. Goud B. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6313-6317Crossref PubMed Scopus (219) Google Scholar, 15de Wit H. Lichtenstein Y. Geuze H. Kelly R.B. van der Sluijs P. Klumperman J. Mol. Biol. Cell. 1999; 10: 4163-4176Crossref PubMed Scopus (53) Google Scholar). rab4 is phosphorylated on Ser-196 during mitosis (16Bailly E. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Crossref PubMed Scopus (130) Google Scholar, 17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). This increases the cytoplasmic pool ∼5–10-fold, possibly due to the interaction with the mitotic peptidyl-prolyl isomerase Pin1 (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Surprisingly, these phosphorylated rab4 molecules are in the GTP form. Because the cytoplasmic pool of rab proteins is thought to be in the GDP-bound form, this suggests that the additional rab4 molecules in the cytoplasm of mitotic cells are dissociated from early endosomes. Perturbation of the normal rab4 cycle by phosphorylation of either the cytoplasmic or membrane-bound pools may result in an increased cytoplasmic steady-state localization during mitosis. Hence, it has been difficult to establish whether membrane-bound rab4 is a target for mitotic kinases. To address this question, and to investigate whether membrane transport through early endosomes depends on cycles of rab4 dissociation and association with early endosomes, we constructed NHrab4cbvn in which we replaced the carboxyl-terminal Cys-Gly-Cys motif of rab4 with the transmembrane domain of cellubrevin, an endosomal v-SNARE. We found that NHrab4cbvn was fixed permanently on endosomal membranes and did not support rab4 function in membrane transport. The chimeric protein was phosphorylated during mitosis, showing that membrane-bound rab4 is targeted by mitotic kinases. DISCUSSIONProtein phosphorylation plays a key role in the regulation of membrane transport and organelle inheritance during the mammalian cell cycle. Intracellular transport is coordinately inhibited in dividing cells, and organelle fragmentation is thought to occur due to ongoing transport vesicle formation while membrane fusion is inhibited. The molecular mechanisms underlying the inhibition of membrane transport during mitosis are only partially understood. rab4 regulates membrane transport through early endosomes and is phosphorylated by p34cdc2 kinase (11van der Sluijs P. Hull M. Webster P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Thus rab4 is likely to be one of the targets for the inhibition of endocytic transport. Indeed, although rab4 is associated with endosomes in interphase, during mitosis it is localized to the cytoplasm in a complex with the peptidyl prolylisomerase Pin1 and presumably unable to perform its endosomal function (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Why phosphorylated rab4 is localized to the cytoplasm is not clear. Phosphorylation of the cytoplasmic rab4 pool might inhibit its recruitment to endosomes. Alternatively, phosphorylation of membrane-bound rab4 may enhance its dissociation into the cytoplasm. Both scenarios or a combination thereof will result in the depletion of rab4 from early endosomes and the build-up of a cytoplasmic pool of rab4 molecules. A rab4 truncation mutant that is constitutively expressed in the cytoplasm does become phosphorylated during mitosis, which is consistent with a model in which phosphorylated cytoplasmic rab4 molecules fail to be recruited to endosomes (17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Informative as it is, this result cannot be used as evidence against the second model, because it is not known whether the endosomal pool of rab4 might be targeted by a mitotic kinase.We addressed this question in expression experiments with an rab4 construct that was permanently attached to early endosomes, because its consensus sequence for carboxyl-terminal isoprenylation was replaced by the transmembrane domain of the endosomal v-SNARE, cellubrevin. The chimeric protein was membrane-associated in both interphase and mitotic cells. During mitosis NHrab4cbvn was phosphorylated, showing for the first time that phosphorylation of rab4 can also occur on membranes. This also explains the previous finding that phosphorylated cytoplasmic rab4 is in the GTP-bound form (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Because rab4 guanine nucleotide exchange activity remains membrane-associated during mitosis (32Ayad N. Hull M. Mellman I. EMBO J. 1997; 16: 4497-4507Crossref PubMed Scopus (48) Google Scholar), enhanced dissociation of phosphorylated rab4-GTP from endosomes increases the number of cytoplasmic rab4 molecules in the GTP-bound form.GTP hydrolysis on rab proteins was initially thought to be required for membrane docking and fusion or recycling of the GDP-bound form back to the donor organelle (39Bourne H.R. Cell. 1988; 53: 669-671Abstract Full Text PDF PubMed Scopus (340) Google Scholar). More recent experiments with Ypt1p and Sec4p that were permanently attached to membranes have called this concept into question (9Ossig R. Laufer W. Schmitt H.D. Gallwitz D. EMBO J. 1995; 14: 3645-3653Crossref PubMed Scopus (32) Google Scholar). These mutants appeared to be targeted to the correct membranes and reportedly retained the capacity to maintain vesicular transport through the biosynthetic pathway. In contrast to these rab proteins that act between the ER and the plasma membrane, our recycling experiments in MDCK cells, however, showed that NHrab4cbvn cannot substitute for wild type rab4.Because rab proteins are important for tethering of transport vesicles to their target membrane (see Ref. 40Brennwald P. J. Cell Biol. 2000; 149: 1-3Crossref PubMed Google Scholar) and for their formation at the donor compartment (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 41Nuoffer C. Davidson H. Matteson J. Meinkoth J. Balch W.E. J. Cell Biol. 1994; 125: 225-238Crossref PubMed Scopus (191) Google Scholar, 42McLaughlan H. Newell J. Morrice N. Osborne A. West M. Smythe E. Curr. Biol. 1998; 8: 34-45Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 43Carroll K.S. Hanna J. Simon I. Krise J. Barbero P. Pfeffer S.R. Science. 2001; 292: 1373-1376Crossref PubMed Scopus (202) Google Scholar), a salvage pathway is required to recycle rab proteins from the acceptor membrane. GDI-mediated extraction followed by cytosolic transfer of a rab·rab GDI complex back to the donor or transport vesicle is an attractive mechanism that is supported by experimental evidence, although it is also possible that retrieval to the donor membrane may occur via a retrograde transport pathway. The observation, that NHrab4cbvn permanently resides on membranes and has an inhibitory phenotype in the transport assay, suggests that a cycle of rab4 between membrane and cytosol is important for its function in membrane recycling from early endosomes. It is unlikely that retrieval of NHrab4cbvn to early endosomes proceeds via the cell surface and endocytosis, because immunoelectron microscopy shows that neither rab4 (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 15de Wit H. Lichtenstein Y. Geuze H. Kelly R.B. van der Sluijs P. Klumperman J. Mol. Biol. Cell. 1999; 10: 4163-4176Crossref PubMed Scopus (53) Google Scholar) nor NHrab4cbvn localize to the plasma membrane.Why would rab4 behave differently in this respect than do Ypt1p and Sec4p? At least two explanations can be put forward to account for this disparity. First, even though the biosynthetic and endocytic pathways are regulated by members of conserved protein families, there appear to be clear differences. For instance, deletion of the YPT1 orSEC4 genes in yeast is lethal (44Segev N. Mullholland J. Botstein D. Cell. 1988; 52: 915-924Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 45Salminen A. Novick P.J. Cell. 1987; 49: 527-538Abstract Full Text PDF PubMed Scopus (589) Google Scholar), whereas knockout strains of YPT5, whose product regulates the endocytic pathway, are viable (46Horazdovski B.F. Busch G.R. Emr S. EMBO J. 1994; 13: 1297-1309Crossref PubMed Scopus (168) Google Scholar). Second, because rab GTPases exert their functions in membrane transport through multiple effector proteins (reviewed in Ref. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar), this may simply indicate that there is not necessarily a common principle by which rab proteins control membrane traffic. An example of this idea is provided by rab27a, which in melanocytes cooperates with myosin Va (6Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar, 47Wu X.S. Rao K. Zhang H. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 1-8Crossref PubMed Scopus (379) Google Scholar) but not in cytotoxic T lymphocytes (48Haddad E.K., Wu, X. Hammer III, J.A. Henkart P.A. J. Cell Biol. 2001; 152: 835-842Crossref PubMed Scopus (208) Google Scholar). Presently, five rab4 effector proteins have been characterized. These are rabaptin-4 (49Nagelkerken B. van Anken E. van Raak M. Gerez L. Mohrmann K. van Uden N. Holthuizen J. Pelkmans L. van der Sluijs P. Biochem. J. 2000; 346: 593-601Crossref PubMed Scopus (55) Google Scholar), rabaptin-5 (50Vitale G. Rybin V. Christoforidis S. Thornqvist P.O. McCaffrey M. Stenmark H. Zerial M. EMBO J. 1998; 17: 1941-1951Crossref PubMed Scopus (195) Google Scholar), rabenosyn-5 (51de Renzis S. Sönnichsen B. Zerial M. Nat. Cell Biol. 2002; 4: 124-133Crossref PubMed Scopus (260) Google Scholar), rabip4 (52Cormont M. Mari M. Galmiche A. Hofman P. Le Marchand-Brustel Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1637-1642Crossref PubMed Scopus (85) Google Scholar), and the cytoplasmic dynein light intermediate chain-1 (53Bielli A. Thörnqvist P.O. Hendrick A.G. Finn R. Fitzgerald K. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2001; 281: 1141-1153Crossref PubMed Scopus (77) Google Scholar), the first three of which are bifunctional ones that also interact with rab5. Clearly a further understanding of membrane recycling via early endosomes requires the identification and characterization of additional rab4 effector and accessory proteins. Protein phosphorylation plays a key role in the regulation of membrane transport and organelle inheritance during the mammalian cell cycle. Intracellular transport is coordinately inhibited in dividing cells, and organelle fragmentation is thought to occur due to ongoing transport vesicle formation while membrane fusion is inhibited. The molecular mechanisms underlying the inhibition of membrane transport during mitosis are only partially understood. rab4 regulates membrane transport through early endosomes and is phosphorylated by p34cdc2 kinase (11van der Sluijs P. Hull M. Webster P. Goud B. Mellman I. Cell. 1992; 70: 729-740Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Thus rab4 is likely to be one of the targets for the inhibition of endocytic transport. Indeed, although rab4 is associated with endosomes in interphase, during mitosis it is localized to the cytoplasm in a complex with the peptidyl prolylisomerase Pin1 and presumably unable to perform its endosomal function (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Why phosphorylated rab4 is localized to the cytoplasm is not clear. Phosphorylation of the cytoplasmic rab4 pool might inhibit its recruitment to endosomes. Alternatively, phosphorylation of membrane-bound rab4 may enhance its dissociation into the cytoplasm. Both scenarios or a combination thereof will result in the depletion of rab4 from early endosomes and the build-up of a cytoplasmic pool of rab4 molecules. A rab4 truncation mutant that is constitutively expressed in the cytoplasm does become phosphorylated during mitosis, which is consistent with a model in which phosphorylated cytoplasmic rab4 molecules fail to be recruited to endosomes (17van der Sluijs P. Hull M. Huber L.A. Male P. Goud B. Mellman I. EMBO J. 1992; 11: 4379-4389Crossref PubMed Scopus (112) Google Scholar). Informative as it is, this result cannot be used as evidence against the second model, because it is not known whether the endosomal pool of rab4 might be targeted by a mitotic kinase. We addressed this question in expression experiments with an rab4 construct that was permanently attached to early endosomes, because its consensus sequence for carboxyl-terminal isoprenylation was replaced by the transmembrane domain of the endosomal v-SNARE, cellubrevin. The chimeric protein was membrane-associated in both interphase and mitotic cells. During mitosis NHrab4cbvn was phosphorylated, showing for the first time that phosphorylation of rab4 can also occur on membranes. This also explains the previous finding that phosphorylated cytoplasmic rab4 is in the GTP-bound form (18Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z., Lu, K.P. van der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Because rab4 guanine nucleotide exchange activity remains membrane-associated during mitosis (32Ayad N. Hull M. Mellman I. EMBO J. 1997; 16: 4497-4507Crossref PubMed Scopus (48) Google Scholar), enhanced dissociation of phosphorylated rab4-GTP from endosomes increases the number of cytoplasmic rab4 molecules in the GTP-bound form. GTP hydrolysis on rab proteins was initially thought to be required for membrane docking and fusion or recycling of the GDP-bound form back to the donor organelle (39Bourne H.R. Cell. 1988; 53: 669-671Abstract Full Text PDF PubMed Scopus (340) Google Scholar). More recent experiments with Ypt1p and Sec4p that were permanently attached to membranes have called this concept into question (9Ossig R. Laufer W. Schmitt H.D. Gallwitz D. EMBO J. 1995; 14: 3645-3653Crossref PubMed Scopus (32) Google Scholar). These mutants appeared to be targeted to the correct membranes and reportedly retained the capacity to maintain vesicular transport through the biosynthetic pathway. In contrast to these rab proteins that act between the ER and the plasma membrane, our recycling experiments in MDCK cells, however, showed that NHrab4cbvn cannot substitute for wild type rab4. Because rab proteins are important for tethering of transport vesicles to their target membrane (see Ref. 40Brennwald P. J. Cell Biol. 2000; 149: 1-3Crossref PubMed Google Scholar) and for their formation at the donor compartment (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 41Nuoffer C. Davidson H. Matteson J. Meinkoth J. Balch W.E. J. Cell Biol. 1994; 125: 225-238Crossref PubMed Scopus (191) Google Scholar, 42McLaughlan H. Newell J. Morrice N. Osborne A. West M. Smythe E. Curr. Biol. 1998; 8: 34-45Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 43Carroll K.S. Hanna J. Simon I. Krise J. Barbero P. Pfeffer S.R. Science. 2001; 292: 1373-1376Crossref PubMed Scopus (202) Google Scholar), a salvage pathway is required to recycle rab proteins from the acceptor membrane. GDI-mediated extraction followed by cytosolic transfer of a rab·rab GDI complex back to the donor or transport vesicle is an attractive mechanism that is supported by experimental evidence, although it is also possible that retrieval to the donor membrane may occur via a retrograde transport pathway. The observation, that NHrab4cbvn permanently resides on membranes and has an inhibitory phenotype in the transport assay, suggests that a cycle of rab4 between membrane and cytosol is important for its function in membrane recycling from early endosomes. It is unlikely that retrieval of NHrab4cbvn to early endosomes proceeds via the cell surface and endocytosis, because immunoelectron microscopy shows that neither rab4 (13de Wit H. Lichtenstein Y. Kelly R.B. Geuze H.J. Klumperman J. van der Sluijs P. Mol. Biol. Cell. 2001; 12: 3703-3715Crossref PubMed Scopus (57) Google Scholar, 15de Wit H. Lichtenstein Y. Geuze H. Kelly R.B. van der Sluijs P. Klumperman J. Mol. Biol. Cell. 1999; 10: 4163-4176Crossref PubMed Scopus (53) Google Scholar) nor NHrab4cbvn localize to the plasma membrane. Why would rab4 behave differently in this respect than do Ypt1p and Sec4p? At least two explanations can be put forward to account for this disparity. First, even though the biosynthetic and endocytic pathways are regulated by members of conserved protein families, there appear to be clear differences. For instance, deletion of the YPT1 orSEC4 genes in yeast is lethal (44Segev N. Mullholland J. Botstein D. Cell. 1988; 52: 915-924Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 45Salminen A. Novick P.J. Cell. 1987; 49: 527-538Abstract Full Text PDF PubMed Scopus (589) Google Scholar), whereas knockout strains of YPT5, whose product regulates the endocytic pathway, are viable (46Horazdovski B.F. Busch G.R. Emr S. EMBO J. 1994; 13: 1297-1309Crossref PubMed Scopus (168) Google Scholar). Second, because rab GTPases exert their functions in membrane transport through multiple effector proteins (reviewed in Ref. 1Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2681) Google Scholar), this may simply indicate that there is not necessarily a common principle by which rab proteins control membrane traffic. An example of this idea is provided by rab27a, which in melanocytes cooperates with myosin Va (6Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar, 47Wu X.S. Rao K. Zhang H. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell Biol. 2002; 4: 1-8Crossref PubMed Scopus (379) Google Scholar) but not in cytotoxic T lymphocytes (48Haddad E.K., Wu, X. Hammer III, J.A. Henkart P.A. J. Cell Biol. 2001; 152: 835-842Crossref PubMed Scopus (208) Google Scholar). Presently, five rab4 effector proteins have been characterized. These are rabaptin-4 (49Nagelkerken B. van Anken E. van Raak M. Gerez L. Mohrmann K. van Uden N. Holthuizen J. Pelkmans L. van der Sluijs P. Biochem. J. 2000; 346: 593-601Crossref PubMed Scopus (55) Google Scholar), rabaptin-5 (50Vitale G. Rybin V. Christoforidis S. Thornqvist P.O. McCaffrey M. Stenmark H. Zerial M. EMBO J. 1998; 17: 1941-1951Crossref PubMed Scopus (195) Google Scholar), rabenosyn-5 (51de Renzis S. Sönnichsen B. Zerial M. Nat. Cell Biol. 2002; 4: 124-133Crossref PubMed Scopus (260) Google Scholar), rabip4 (52Cormont M. Mari M. Galmiche A. Hofman P. Le Marchand-Brustel Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1637-1642Crossref PubMed Scopus (85) Google Scholar), and the cytoplasmic dynein light intermediate chain-1 (53Bielli A. Thörnqvist P.O. Hendrick A.G. Finn R. Fitzgerald K. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2001; 281: 1141-1153Crossref PubMed Scopus (77) Google Scholar), the first three of which are bifunctional ones that also interact with rab5. Clearly a further understanding of membrane recycling via early endosomes requires the identification and characterization of additional rab4 effector and accessory proteins. We thank Thomas Südhof and Thierry Galli and the late Thomas Kreis for generously sharing reagents and our colleagues at the Department of Cell Biology for helpful suggestions. We also thank Rene Scriwanek and Marc van Peski for artwork and photography.
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