Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe.

The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.     

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.     

Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.     

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.     

Rab 7: an important regulator of late endocytic membrane traffic

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

Regulation of endocytosis by the small GTP-ase Rab5

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.     

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.     

Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments

A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.     

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.     

Mutational analysis of the putative effector domain of the GTP-binding Ypt1 protein in yeast suggests specific regulation by a novel GAP activity.

Ypt1p of Saccharomyces cerevisiae is a ras-related GTP-binding protein that fulfils an essential function in intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi complex. Ypt proteins from yeasts and mammals that share an identical sequence in the region analogous to the ras effector domain are functionally interchangeable. We analyzed the function of the putative effector domain of yeast Ypt1p (amino acids 37-45) using site-directed mutagenesis and gene replacement. Four out of six point mutations leading to single amino acid substitutions (Y37F, S39A, T40S and V43E) did not cause any particular phenotype. ypt1(I41M) mutants were inviable whereas ypt1(D44N) mutant cells were temperature sensitive at 37 degrees C and accumulated core-glycosylated invertase at the nonpermissive temperature. This mutant also accumulated ER and small vesicles both at 25 degrees C and 37 degrees C. From porcine liver we identified and partially purified a GTPase-activating protein (yptGAP) that is similarly active with mouse ypt1p/rab1p and yeast Ypt1p but is inactive with H-ras protein as a substrate. Although none of the yeast ypt1 mutant proteins were significantly impaired in their ability to bind GTP, purified ypt1(D44N)p responded only partially and ypt1(I41M)p did not respond at all, to yptGAP. Thus we suggest that analogous to rasGAP/H-ras p21 interaction in mammalian cells, yptGAP is an intracellular target of Ypt1p, interacting with the effector domain and regulating its GTPase activity, and that this interaction is required for the functioning of yeast Ypt1p in intracellular protein transport.     

Rab4 Regulates Formation of Synaptic-like Microvesicles from Early Endosomes in PC12 Cells

Early endosomes in PC12 cells are an important site for the formation of synaptic-like microvesicles and constitutive recycling vesicles. By immunogold electron microscopy, the small GTPase rab4 was localized to early endosomes and numerous small vesicles in the cell periphery and Golgi area of PC12 cells. Overexpression of GTPase-deficient Q67Lrab4 increased the number of early endosome-associated and cytoplasmic vesicles, whereas expression of GDP-bound S22Nrab4 significantly increased the length of early endosomal tubules. In parallel, Q67Lrab4 induced a shift in rab4, VAMP2, and TfR label from early endosomes to peripheral vesicles, whereas S22Nrab4 increased early endosome labeling of all three proteins. These observations were corroborated by early endosome budding assays. Together, our data document a thus far unrecognized role for rab4 in the formation of synaptic-like microvesicles and add to our understanding of the formation of constitutive recycling vesicles from early endosomes.     

The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.     

Identification of ras-related, YPT family genes in Schizosaccharomyces pombe.

Screening for genes homologous to ras in Schizosaccharomyces pombe resulted in the isolation of a homolog of Saccharomyces cerevisiae YPT1. This S. pombe gene, named ypt3, has a coding capacity of 214 amino acids interrupted by two introns, and is essential for cell growth. Two more YPT1 homologs were isolated from S. pombe using a part of the ypt3 gene as the probe. One of them, named ypt1, is highly homologous to S. cerevisiae YPT1 and mouse ypt1 and is essential for cell growth. This gene has four introns and encodes 203 amino acids. Its cDNA placed downstream of the S. cerevisiae GAL7 promoter could complement S. cerevisiae ypt1-, indicating that Sp ypt1 and Sc YPT1 are functionally homologous. The other isolate, named ryh1, and a fourth homolog, ypt2, have been characterized by Gallwitz and co-workers. The ypt1, ypt2 and ypt3 genes, but not ryh1, constitute a family, their products having double cysteine as their C terminus and serine in place of a glycine residue highly conserved in ras proteins (mammalian Gly-12 or S. pombe Gly-17). The physiological roles of these genes appear to be distinct because each of them is indispensable for cell growth.     

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

rab5 controls early endosome fusion in vitro

The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.     

Distinct structural elements of rab5 define its functional specificity.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.     

Direct activation of the fission yeast PAK Shk1 by the novel SH3 domain protein, Skb5

The p21-activated kinase (PAK) homolog Shk1 is essential for cell viability in the fission yeast Schizosaccharomyces pombe. Roles have been established for Shk1 in the regulation of cell morphology, sexual differentiation, and mitosis in S. pombe. In this report, we describe the genetic and molecular characterization of a novel SH3 domain protein, Skb5, identified as a result of a two-hybrid screen for Shk1 interacting proteins. S. pombe cells carrying a deletion of the skb5 gene exhibit no discernible phenotypic defects under normal growth conditions, but when subjected to hypertonic stress, become spheroidal in shape and growth impaired. Both of these defects can be suppressed by overexpression of the Shk1 modulator, Skb1. The growth inhibition that results from overexpression of Shk1 in S. pombe cells is markedly suppressed by a null mutation in the skb5 gene, suggesting that Skb5 contributes positively to the function of Shk1 in vivo. Consistent with this notion, we show that Skb5 stimulates Shk1 catalytic function in S. pombe cells. Furthermore, and perhaps most significantly, we show that bacterially expressed recombinant Skb5 protein directly stimulates the catalytic activity of recombinant Shk1 kinase in vitro. These and additional data described herein demonstrate that Skb5 is a direct activator of Shk1 in fission yeast.     

Two Fission Yeast Rab7 Homologs, Ypt7 and Ypt71, Play Antagonistic Roles in the Regulation of Vacuolar Morphology

Small guanine triphosphatases (GTPases) of the Rab family are key regulators of membrane trafficking events between the various subcellular compartments in eukaryotic cells. Rab7 is a conserved protein required in the late endocytic pathway and in lysosome biogenesis. A Schizosaccharomyces pombe ( S. pombe ) homolog of Rab7, Ypt7, is necessary for trafficking from the endosome to the vacuole and for homotypic vacuole fusion. Here, we identified and characterized a second fission yeast Rab7 homolog, Ypt71. Ypt71 is localized to the vacuolar membrane. Cells deleted for ypt71 ⁺ exhibit normal growth rates and morphology. Interestingly, a ypt71 null mutant contains large vacuoles in contrast with the small fragmented vacuoles found in the ypt7 null mutant. Furthermore, the ypt71 mutation does not enhance or alleviate the temperature sensitivity or vacuole fusion defect of ypt7 Δ cells. Like ypt7 Δ cells, overexpression of ypt71 ⁺ caused fragmentation of vacuoles and inhibits vacuole fusion under hypotonic conditions. Thus, the two S. pombe Rab7 homologs act antagonistically in regulating vacuolar morphology. Analysis of a chimeric Ypt7/Ypt71 protein showed that Rab7-directed vacuole dynamics, fusion versus fission, largely depends on the medial region of the protein, including a part of RabSF3/α3-L7.