The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.

We have investigated the in vivo functional role of rab5, a small GTPase associated with the plasma membrane and early endosomes. Wild-type rab5 or rab5-ile133, a mutant protein defective in GTP binding, was overexpressed in baby hamster kidney cells. In cells expressing the rab5ile 133 protein, the rate of endocytosis was decreased by 50% compared with normal, while the rate of recycling was not significantly affected. The morphology of early endosomes was also drastically changed by the mutant protein, which induced accumulation of small tubules and vesicles at the periphery of the cell. Surprisingly, overexpression of wild-type rab5 accelerated the uptake of endocytic markers and led to the appearance of atypically large early endosomes. We conclude that rab5 is a rate-limiting component of the machinery regulating the kinetics of membrane traffic in the early endocytic pathway.     

The small GTP-binding protein rab6 functions in intra-Golgi transport

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Geraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha- mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.     

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.     

The roles of ubiquitin and lipids in protein sorting along the endocytic pathway

After cell surface receptors are internalized for endocytosis, they are accurately sorted in endosomes. Some are recycled to the plasma membrane and others are downregulated by delivery to lysosomes. Evidence is rapidly accumulating that ubiquitination of cargo proteins acts as a sorting signal during endocytosis. Sorting devices that recognize ubiquitin are distributed to various compartments, probably acting in a concerted manner. Cholesterol is enriched in the plasma membrane and endosomes, and is involved in protein sorting by forming microdomains called lipid rafts. Ubiquitin and cholesterol hold the key to control the endocytic sorting, and they are likely acting cooperatively.     

The RCP-Rab11 complex regulates endocytic protein sorting

Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.     

Rab15 mediates an early endocytic event in Chinese hamster ovary cells.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.     

Characterization of a small GTP-binding protein of the rab 5 family in Mesembryanthemum crystallinum with increased level of expression during early salt stress

A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b were only found in plants and not in yeast or vertebrates suggests that they have plant-specific functions. The expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress. After heterologous expression in Escherichia coli an antibody was raised against recombinant McRAB5b protein. Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP, while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition.     

COPI-mediated protein and lipid sorting in the early secretory pathway

Summary The major function of the secretory pathway of eukaryotes is to maintain the compartmental organization of the endomembrane system and organelle-associated functions by proper distribution of newly synthesized molecules. Protein and lipid transport is mediated by vesicular intermediates that connect the various organelles throughout this pathway. This principle enables the eukaryotic cell to actively sort proteins and lipids at every level of this route, in both the anterograde and the retrograde direction. Here, we discuss the molecular mechanisms of nonclathrin (COPI and COPII)-coated-vesicle biogenesis and how transport vesicle formation is linked to protein and lipid sorting in the early secretory pathway.     

The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease

Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH(4)(+), a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(ts) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH(4)(+) is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH(4)(+). Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.     

Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum

To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine- and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments.     

The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway

SAR1, the yeast gene which encodes a novel type of small GTP-binding protein, has been shown to be required for protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To further the understanding of the function of its product, a lacZ-SAR1 hybrid gene was constructed and a polyclonal antibody was raised against the hybrid protein. This antibody specifically recognizes the SAR1 gene product (Sar1p) as a 23-kDa protein in the yeast cell lysate. We examined the subcellular localization of Sar1p using this antibody. In wild-type cells, Sar1p was predominantly recovered in a rapidly sedimenting membrane fraction that includes the ER. The soluble form of Sar1p was also detected when the protein was overproduced. Immunofluorescence microscopy with the anti-Sar1p antibody showed perinuclear staining that was exaggerated in the ER-accumulating sec18 mutant. Membrane association of Sar1p was shown to be very light. Sar1p was not extracted from the membrane by treatment with alkaline sodium carbonate, and only 1% deoxycholic acid solubilized Sar1p completely. From these results, we suggest that Sar1p is firmly located on the ER membrane where it regulates the ER-Golgi traffic.     

Rabip4' is an effector of rab5 and rab4 and regulates transport through early endosomes

We describe the characterization of an 80-kDa protein cross-reacting with a monoclonal antibody against the human La autoantigen. The 80-kDa protein is a variant of rabip4 with an N-terminal extension of 108 amino acids and is expressed in the same cells. For this reason, we named it rabip4'. rabip4' is a peripheral membrane protein, which colocalized with internalized transferrin and EEA1 on early endosomes. Membrane association required the presence of the FYVE domain and was perturbed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Expression of a dominant negative rabip4' mutant reduced internalization and recycling of transferrin from early endosomes, suggesting that it may be functionally linked to rab4 and rab5. In agreement with this, we found that rabip4' colocalized with the two GTPases on early endosomes and bound specifically and simultaneously to the GTP form of both rab4 and rab5. We conclude that rabip4' may coordinate the activities of rab4 and rab5, regulating membrane dynamics in the early endosomal system.     

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.     

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.     

The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system

A cell-free system which reconstitutes early stages of receptor-mediated endocytosis has been developed, based on detection of the association between avidin-beta-galactosidase (Av beta Gal) and biotin-transferrin (B-Tf). Initially, Av beta Gal (a fluid-phase marker) and B-Tf (receptor-bound) are internalized and delivered to a common endosomal compartment in vivo and in vitro. Subsequently, these two probes enter divergent intracellular pathways: Av beta Gal is sorted from the endosome and directed for delivery to lysosomes, whereas B-Tf is segregated away from the fluid-phase marker, remaining bound to the transferrin receptor for return to the cell surface. Using the avidin-biotin association reaction to monitor the co-localization of these two probes, we have been able to reconstruct this sorting and segregation process in a cell-free system. The in vitro reaction is time-, temperature-, and ATP-dependent, and is not affected by NH4Cl; cell-free segregation of the two probes is also sensitive to N-ethylmaleimide. As these characteristics are also properties of in vitro endocytic vesicle fusion, it is likely that the latter event is a prerequisite for the sorting and segregation process. Both the in vivo and in vitro sorting of Av beta Gal and B-Tf to their respective and distinct destinations can be followed by subcellular fractionation on Percoll gradients. Our observations provide the first evidence that the cellular mechanism to identify, sort, and sequester endocytosed material can be reconstituted in a cell-free system.     

rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

rab3A is a low molecular weight (LMW) GTP-binding protein specifically expressed in brain and localized to synaptic vesicles. rab3A has been proposed to play a role in neurotransmitter release by regulating membrane flow in the nerve terminal. In an attempt to define other LMW GTP-binding proteins that may regulate neurotransmitter release, seven cDNA clones encoding new members of the rab family of LMW GTP-binding proteins were isolated from a rat brain cDNA library. The rab proteins contain the four conserved structural domains essential for GTP binding in addition to domains required for membrane localization and effector protein interactions. One protein, rab16, is closely related to members of the rab3 subfamily, whereas two others are assigned as the rat homologs of canine rab8 and rab10. Four additional clones, rab12, rab13, rab14, and rab15, revealed unique sequences and are new members of the rab family of LMW GTP-binding proteins. The patterns of expression of rab15 and rab3A closely overlap but differ from that observed for all other known LMW GTP-binding proteins. This data suggests that rab15 may act in concert with rab3A in regulating aspects of synaptic vesicle membrane flow within the nerve terminal.     

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P(2) and PI(3,4,5)P(3), at the cell surface.     

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.