Iris Simón

PURPOSE: This study aims to develop a robust gene expression classifier that can predict disease relapse in patients with early-stage colorectal cancer (CRC). PATIENTS AND METHODS: Fresh frozen tumor tissue from 188 patients with stage I to IV CRC undergoing surgery was analyzed using Agilent 44K oligonucleotide arrays. Median follow-up time was 65.1 months, and the majority of patients (83.6%) did not receive adjuvant chemotherapy. A nearest mean classifier was developed using a cross-validation procedure to score all genes for their association with 5-year distant metastasis-free survival. RESULTS: An optimal set of 18 genes was identified and used to construct a prognostic classifier (ColoPrint). The signature was validated on an independent set of 206 samples from patients with stage I, II, and III CRC. The signature classified 60% of patients as low risk and 40% as high risk. Five-year relapse-free survival rates were 87.6% (95% CI, 81.5% to 93.7%) and 67.2% (95% CI, 55.4% to 79.0%) for low- and high-risk patients, respectively, with a hazard ratio (HR) of 2.5 (95% CI, 1.33 to 4.73; P = .005). In multivariate analysis, the signature remained one of the most significant prognostic factors, with an HR of 2.69 (95% CI, 1.41 to 5.14; P = .003). In patients with stage II CRC, the signature had an HR of 3.34 (P = .017) and was superior to American Society of Clinical Oncology criteria in assessing the risk of cancer recurrence without prescreening for microsatellite instability (MSI). CONCLUSION: ColoPrint significantly improves the prognostic accuracy of pathologic factors and MSI in patients with stage II and III CRC and facilitates the identification of patients with stage II disease who may be safely managed without chemotherapy.

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

Rab GTPases represent a large family of Ras-like enzymes that play key roles in the secretory and endocytic pathways. They are located on distinct membrane-bound compartments, and genetic experiments implicate Rabs in the processes by which transport vesicles or membrane-bound compartments recognize their cognate fusion targets (see Refs. 1Novick P. Brennwald P. Friends and family: the role of Rab GTPases in vesicular traffic..Cell. 1993; 75: 597-601Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 2Nuoffer C. Balch W.E. GTPases: multifunctional molecular switches regulating vesicular traffic.Annu Rev. Biochem. 1994; 63: 949-990Crossref PubMed Scopus (374) Google Scholar, 3Pfeffer S.R. Rab GTPases: master regulators of membrane trafficking..Curr. Biol. 1994; 6: 522-526Crossref Scopus (296) Google Scholar, 4Novick P. Zerial M. The diversity of Rab proteins in vesicle transport..Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (661) Google Scholar for review). Because mutant forms of Rab proteins can block protein transport along a given route or actually change the sizes of entire organelles, Rabs obviously play key regulatory roles in membrane trafficking. This minireview will attempt to summarize our current view of what Rabs do. Most Rabs are doubly geranylgeranylated at or near their C termini, which leads to their membrane association. The specificity of Rab localization is provided by structural determinants unique to each family member (5Chavrier P. Gorvel J.-P. Stelzer E. Simons K. Gruenberg J. Zerial M. Hypervariable C-terminal domain of Rab proteins acts as a targeting signal..Nature. 1991; 353: 769-772Crossref PubMed Scopus (318) Google Scholar, 6Brennwald P. Novick P. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4.Nature. 1993; 362: 560-563Crossref PubMed Scopus (154) Google Scholar, 7Dunn B. Stearns T. Botstein D. Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4..Nature. 1993; 362: 563-565Crossref PubMed Scopus (90) Google Scholar, 8Stenmark H. Valencia A. Martinez O. Ullrich O. Goud B. Zerial M. Distinct structural elements of Rab5 define its functional specificity..EMBO J. 1994; 13: 575-583Crossref PubMed Scopus (146) Google Scholar) that appear to be recognized by distinct sets of proteins on organelle surfaces (9Soldati T. Shapiro A.D. Dirac-Svejstrup A.B. Pfeffer S.R. Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange..Nature. 1994; 369: 76-78Crossref PubMed Scopus (151) Google Scholar, 10Ullrich O. Horiuchi H. Bucci C. Zerial M. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange..Nature. 1994; 368: 157-160Crossref PubMed Scopus (249) Google Scholar, 11Dirac-Svejstrup A.B. Sumizawa D. Pfeffer S.R. Identification of an endosomal GDI displacement factor that displaces prenylated Rab GTPases from Rab-GDI..EMBO J. 1997; 16: 465-472Crossref PubMed Scopus (169) Google Scholar, 12Ayad N. Hull M. Mellman I. Mitotic phosphorylation of Rab4 prevents binding to a specific receptor on endosome membranes..EMBO J. 1997; 16: 4497-4507Crossref PubMed Scopus (48) Google Scholar). Like Ras, Rabs cycle between an active, GTP-bound form and an inactive, GDP-bound form. Transport vesicles carry Rab proteins with bound GTP; after membrane fusion, GTP hydrolysis converts them into their GDP-bound states. A cytosolic protein, termed GDI, retrieves prenylated, GDP-bound Rab proteins from their fusion targets and recycles them back to their membranes of origin. GDI delivers Rabs to membranes with GDP bound; they are subsequently reactivated by Rab-specific, nucleotide exchange factors (13Walch-Solimena C. Collins R.N. Novick P.J. Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles..J. Cell Biol. 1997; 137: 1495-1509Crossref PubMed Scopus (270) Google Scholar, 14Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function..Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). At steady state, the bulk of a given Rab is membrane-associated; however, between 10 and 50% can be detected in the cytosol. soluble N-ethylmaleimide-sensitive factor receptor vesicle SNARE target SNARE endoplasmic reticulum. soluble N-ethylmaleimide-sensitive factor receptor vesicle SNARE target SNARE endoplasmic reticulum. Proteins of the VAMP (v-SNARE) and syntaxin (t-SNARE) families are present on transport vesicles and their targets, respectively, and the pairing of cognate of v- and t-SNAREs is believed to provide the specificity of membrane fusion reactions (15Hay J.C. Scheller R.H. SNAREs and NSF in targeted membrane fusion..Curr. Opin. Cell Biol. 1997; 9: 505-512Crossref PubMed Scopus (254) Google Scholar). Genetic experiments in yeast first linked Rab function to SNARE proteins. Although the yeast ER-to-Golgi Rab, Ypt1p, is an essential gene product, a number of proteins that function as SNARE components in this transport reaction can compensate for the loss of Ypt1p when overexpressed (16Dascher C. Ossig R. Gallwitz D. Schmitt H.D. Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily..Mol. Cell. Biol. 1991; 11: 872-885Crossref PubMed Scopus (279) Google Scholar, 17Lian J.P. Stone S. Jiang Y. Lyons P. Ferro-Novick S. Ypt1p implicated in v-SNARE activation..Nature. 1994; 372: 698-701Crossref PubMed Scopus (161) Google Scholar). Similarly, Brennwald et al. (18Brennwald P. Kearns B. Champion K. Keränen S. Bankaitis V. Novick P. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis..Cell. 1994; 79: 245-258Abstract Full Text PDF PubMed Scopus (311) Google Scholar) found that a t-SNARE homolog could suppress an effector domain mutation in the secretory vesicle Rab, Sec4p. Overexpression of a SNARE could override the need for a protein that facilitates SNARE pairing by simple mass action. Subsequent work confirmed that Ypt1p is required for actual v-SNARE·t-SNARE complex formation (19Søgaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Söllner T. A Rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles..Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 20Lupashin V.V. Waters M.G. t-SNARE activation through transient interaction with a Rab-like guanosine triphosphatase..Science. 1997; 276: 1255-1258Crossref PubMed Scopus (183) Google Scholar). The SNARE complexes analyzed by these workers contained the v- and t-SNAREs, Bos1p and Sed5p, but not the corresponding Rab protein. Thus, Rabs act to facilitate SNARE complex formation but are not core elements of such complexes. If cognate v- and t-SNAREs could pair at all times, all of the organelles in the cytoplasm might become stuck together as part of a giant sandwich. For example, an ER transport vesicle v-SNARE, which resides in the ER, would bind tightly to a Golgi t-SNARE and thereby attach together two entire organelles. Similarly, a post-Golgi v-SNARE would bind the Golgi to a plasma membrane-localized t-SNARE. For this reason, sets of proteins ("SNARE protectors") must block SNARE accessibility. Sec1p is the prototypic t-SNARE-protector. The mammalian homolog of yeast Sec1p (also named n-Sec1, munc18, or rbSec1) binds directly to the presynaptic plasma membrane t-SNARE, syntaxin-1A, with an apparent binding constant in the nanomolar range (21Pevsner J. Hsu S-C. Braun J.E.A. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Specificity and regulation of a synaptic vesicle docking complex..Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar); moreover, Sec1p binding was shown to inhibit v-SNARE·t-SNARE association (21Pevsner J. Hsu S-C. Braun J.E.A. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Specificity and regulation of a synaptic vesicle docking complex..Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar). If t-SNAREs are protected and thus not available for v-SNARE interaction, an additional layer of specificity must be provided to help a vesicle or membrane bearing an active v-SNARE recognize its cognate t-SNARE. We propose that Rabs, in their GTP-bound conformations, recruit transport step-specific docking factors from the cytosol that facilitate v-SNARE·t-SNARE pairing (Fig.1). Such docking factors would be predicted to catalyze the deprotection of t-SNAREs. If the initial Rab recruitment onto a nascent transport vesicle is coupled to (or quickly followed by) its conversion to Rab-GTP, only functional transport vesicles will recruit the docking factors. In this manner, docking will only take place between transport vesicles and their targets, rather than between entire organelles (22Pfeffer S.R. Transport vesicle docking: SNAREs and associates.Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (179) Google Scholar). In contrast, when homotypic fusion between two organelles is the goal, reaction rates would be modulated by the level of Rab-GTP (23Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.G. Goody R. Zerial M. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion..Nature. 1997; 383: 266-269Crossref Scopus (267) Google Scholar). Three macromolecular complexes have been identified that are likely to serve as docking factors: the Exocyst (24TerBush D.R. Novick P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae.J. Cell Biol. 1995; 130: 299-312Crossref PubMed Scopus (252) Google Scholar, 25TerBush D.R. Maurice T. Roth D. Novick P. The exocyst is a multiprotein complex required for exocytosis Saccharomyces cerevisiae.EMBO J. 1996; 15: 6483-6494Crossref PubMed Scopus (678) Google Scholar), the Rabaptin-5 complex (14Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function..Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 26Stenmark H. Vitale G. Ullrich O. Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion..Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar), and another large complex required for ER-to-Golgi transport in yeast (27Sacher M. Jing Y. Barrowman J. Scarpa A. Burston J. Zhang L. Schieltz D. Yates J. Abeliovich H. Ferro Novick S. TRAPP: a highly conserved novel complex on the cis Golgi that mediates vesicle docking and fusion..EMBO J. 1998; 17: 2494-2503Crossref PubMed Scopus (235) Google Scholar). According to our model, such complexes would be recruited onto membranes by a Rab-GTP to help link or direct vesicles to their targets. The Exocyst is a large, 19.5 S particle complex that contains Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo70P and is required for exocytosis. A spectacular finding is the fact that this large "Exocyst" complex is localized to the tips of yeast buds, the site of exocytosis in Saccharomyces cerevisiae. Sec3p appears to localize the Exocyst to the plasma membrane target (28Finger F.P. Hughes T.E. Novick P. Sec3p is a spatial landmark for polarized secretion..Cell. 1998; 92: 559-571Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In contrast, the corresponding t-SNAREs are distributed uniformly over the plasma membrane (18Brennwald P. Kearns B. Champion K. Keränen S. Bankaitis V. Novick P. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis..Cell. 1994; 79: 245-258Abstract Full Text PDF PubMed Scopus (311) Google Scholar). It seems likely, then, that the Exocyst functions to deliver transport vesicles to the docking site on the plasma membrane for the ultimate SNARE assembly reaction. How does a vesicle find the complex? Genetic and biochemical experiments suggest a link between the Sec4p Rab and Exocyst subunits. The data imply that Rabs on a transport vesicle may bind to the Exocyst to position the vesicle for subsequent fusion. Either the docking complex or the Rab (20Lupashin V.V. Waters M.G. t-SNARE activation through transient interaction with a Rab-like guanosine triphosphatase..Science. 1997; 276: 1255-1258Crossref PubMed Scopus (183) Google Scholar) could then free the t-SNARE, allowing for subsequent fusion. In this process, the Exocyst would function as a true "docking" machine. Another docking complex is the Rabaptin-5 complex that interacts with Rab5-GTP in preference to Rab5-GDP (26Stenmark H. Vitale G. Ullrich O. Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion..Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar). Rabaptin-5 is contained within a complex comprised of several subunits, including a nucleotide exchange factor (Rabex-5) that can activate Rab5 (14Horiuchi H. Lippe R. McBride H.M. Rubino M. Woodman P. Stenmark H. Rybin V. Wilm M. Ashman K. Mann M. Zerial M. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function..Cell. 1997; 90: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Interestingly, Rabex-5 activates Rab5, which would further promote Rab5-Rabaptin association. Presumably, while the Rab5·GTP·Rabaptin-5 complex exists, fusion is promoted. Rab5-bound Rabaptin will be in equilibrium with an unbound form. One could imagine that upon Rabaptin-5 release, Rab5-GTP would be instantly hydrolyzed (23Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.G. Goody R. Zerial M. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion..Nature. 1997; 383: 266-269Crossref Scopus (267) Google Scholar). Subsequent Rabex-5 interaction would restore activity to the endosome-associated Rab5. Thus, the Rabaptin-5 complex contains self-regulatory elements that have the capacity to modulate the extent to which this complex is likely to facilitate SNARE pairing in endosome fusion reactions. Expression of a mutant Rab5 that is significantly slowed in its GTP hydrolysis rate triggers the formation of oversized endosomes (29Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. Inhibition of Rab5 GTPase activity stimulates membrane fusion in endocytosis..EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (768) Google Scholar). Because endosome fusion still takes place, these experiments showed elegantly that GTP hydrolysis is not required for nor directly coupled to the membrane fusion event. Even more striking was the recent demonstration that Rab5 hydrolyzes GTP in a continuous cycle that is completely uncoupled to membrane fusion (23Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.G. Goody R. Zerial M. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion..Nature. 1997; 383: 266-269Crossref Scopus (267) Google Scholar). In this study, the authors engineered a Rab5 mutant protein that prefers xanthosine triphosphate to GTP; this mutant enabled them to monitor Rab5-specific nucleotide hydrolysis during an endosome fusion reaction. Zerial and co-workers (23Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.G. Goody R. Zerial M. GTPase activity of Rab5 acts as a timer for endocytic membrane fusion..Nature. 1997; 383: 266-269Crossref Scopus (267) Google Scholar) concluded that the rate of nucleotide hydrolysis by Rabs is rate-determining for membrane docking and fusion reactions. They proposed that cells could regulate the extent of fusion reactions by regulating the steady state level of Rab5-GTP via nucleotide exchange rate-enhancing factors and GTPase-activating proteins. The data showing that Rab5 acts as a timer for endosome fusion are rigorous and convincing. In homotypic endosome-endosome fusion, cells seem to rely upon rapid inactivation of Rab5 to maintain the size of the endosome compartment and to prevent the formation of large vacuoles (29Stenmark H. Parton R.G. Steele-Mortimer O. Lütcke A. Gruenberg J. Zerial M. Inhibition of Rab5 GTPase activity stimulates membrane fusion in endocytosis..EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (768) Google Scholar). In this regard, it is interesting that Rab5 is one of the fastest Rabs in terms of its intrinsic GTPase hydrolysis rate. However, it is important to note that the goal of a homotypic fusion process is rather different from that of a heterotypic vesicle fusion event in which a transport vesicle buds from one compartment and delivers its cargo to the next. In heterotypic processes, it makes little sense to inactivate a Rab prior to vesicle docking and fusion; once a vesicle forms, it should find its target and fuse. In this case,vesicle formation should be rate-limiting, and as described below, Rabs may influence this process as well. To ensure that Rabs remain active on transport vesicles, the transport machinery may make use of a set of Rab-interacting proteins known to lock Rabs in their active conformations. Rabaptin-5 (26Stenmark H. Vitale G. Ullrich O. Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion..Cell. 1995; 83: 423-432Abstract Full Text PDF PubMed Scopus (399) Google Scholar), p40 (30Diaz E. Schimmöller F. Pfeffer S.R. A novel Rab9 effector required for transport from endosomes to the TGN..J. Cell Biol. 1997; 138: 283-290Crossref PubMed Scopus (115) Google Scholar), and Rabphilin (31Shirataki H. Kaibuchi K. Sakoda T. Kishida S. Yamaguchi T. Wada K. Miyazaki M. Takai Y. Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin..Mol. Cell. Biol. 1993; 13: 2061-2068Crossref PubMed Scopus (354) Google Scholar) are all examples of proteins that bind Rabs preferentially in their GTP-bound conformations and concomitantly slow the hydrolysis of GTP by their cognate Rabs. Other Rab effectors such as Rim-3 (32Wang Y. Okamoto M. Schmitz F. Hofmann K. Südhof T.C. Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion..Nature. 1997; 388: 593-598Crossref PubMed Scopus (534) Google Scholar) may play a similar role. Although some of these proteins may bind transiently to drive membrane fusion followed by rapid GTPase inactivation (Rabaptin-5), others may serve as GTP clamps to ensure that membrane fusion takes place before GTP hydrolysis can occur. As described below, such effector proteins are likely to have additional, primary functions in vesicle docking. Whether or not Rabs are rapidly inactivated (as in endosome fusion) or clamped in their GTP-bound conformations, we propose that their primary role is to recruit cytosolic docking factors that are needed for membrane docking and fusion. Several lines of evidence suggest a link between the Rab-mediated process of vesicle docking and the actin- and microtubule-based cytoskeletons (cf. Refs. 13Walch-Solimena C. Collins R.N. Novick P.J. Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles..J. Cell Biol. 1997; 137: 1495-1509Crossref PubMed Scopus (270) Google Scholar and 33Peranen J. Auvinen P. Virta H. Wepf R. Simons K. Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts..J. Cell Biol. 1996; 135: 153-167Crossref PubMed Scopus (214) Google Scholar). An unexpected link was identified for Rab6, which in its GTP-bound conformation interacts with a novel, kinesin-like motor protein named Rabkinesin-6 (34Echard A. Jollivet F. Martinez O. Lacapere J.J. Rousselet A. Janoueix-Lerosey I. Goud B. Interaction of a Golgi-associated kinesin-like protein with Rab6..Science. 1998; 279: 580-585Crossref PubMed Scopus (410) Google Scholar). Rabkinesin-6 is localized to the Golgi complex and may be part of a larger docking complex that uses the microtubule-based cytoskeleton to direct vesicle trafficking. Rabphilin, a Rab3A effector, interacts with the actin-bundling protein, α-actinin, in the absence of Rab3A-GTP (35Kato M. Sasaki T. Ohya T. Nakanishi H. Nishioka H. Imamura M. Takai Y. Physical and functional interaction of rabphilin-3A with α-actinin..J. Biol. Chem. 1996; 271: 31775-31778Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This may provide a means for Rab3A-GTP to modulate the organization of the local actin-based cytoskeleton in relation to events preceding synaptic vesicle exocytosis. Current models for Rab function come in large part from the phenotype of sec4 mutant strains that accumulate secretory vesicles at the non-permissive temperature. This provides strong evidence that Rabs are needed for vesicle docking. Nevertheless, there is accumulating evidence that Rabs must be present in a particular conformation on nascent, budding transport vesicles to permit those vesicles to form (3Pfeffer S.R. Rab GTPases: master regulators of membrane trafficking..Curr. Biol. 1994; 6: 522-526Crossref Scopus (296) Google Scholar). This does not necessarily mean that Rabs function in vesicle budding per se; rather, it is possible that vesicle formation is regulated such that budding only occurs if the vesicle contains everything it needs for docking and fusion. Indirect clues for a requirement for Rabs in vesicle budding came from two studies looking at Rab mutants that bind GDP>GTP. Nuoffer et al. (36Nuoffer C. Davidson H.W. Matteson J. Meinkoth J. Balch W.E. A GDP-bound form of Rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments..J. Cell Biol. 1994; 125: 225-237Crossref PubMed Scopus (192) Google Scholar) found that ER-to-Golgi export was inhibited in the presence of this class of Rab1a mutant. Similar results were obtained by Riederer et al. (37Riederer M.A. Soldati T. Shapiro A.D. Lin J. Pfeffer S.R. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network..J. Cell Biol. 1994; 125: 573-582Crossref PubMed Scopus (244) Google Scholar) studying the role of Rab9 in mannose 6-phosphate receptor trafficking. A Rab9 mutant that bound GDP>GTP did not lead to the accumulation of mannose 6-phosphate receptors in transport vesicles; rather these receptors traveled along other transport routes. In both of these sets of experiments, the mutant protein could have blocked vesicle budding indirectly by sequestering another component required for a downstream process. Very recently, Smythe and co-workers (38McLauchlan H. Newell J. Morrice N. Osborne A. West M. Smythe E. A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits..Curr. Biol. 1998; 8: 34-45Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) showed directly that Rab5 is required for ligand sequestration into clathrin-coated pits. Given the well established role of Rab5 in the fusion of endocytic vesicles with endosomes, these data support the proposal that the recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles. If Rabs are needed for vesicle formation, why would vesicles accumulate in a sec4 mutant strain? Perhaps the budding machinery can sense the presence of a Rab in its GTP-bound conformation. Thus, certain mutant Rab alleles could be incorporated into a vesicle budding machine but not be able to function in docking, other mutant alleles could a Rab that blocked vesicle formation as well. yeast proteins have functions in when is cells accumulate Golgi in strains bearing a M. F. Gallwitz D. GTPase and are essential for Golgi function in J. 1996; 15: PubMed Scopus Google Scholar, G. J. N. GTPases are required for from the yeast trans-Golgi Cell Biol. 1997; 137: PubMed Scopus (179) Google Scholar). This phenotype could be if this be recognized by the vesicle budding In cells have to a of to at Rab with distinct and unique roles in the secretory and endocytic pathways. of these proteins are essential for in if they their functions are by Rab Thus, Rabs are key regulators of membrane reactions.