Small GTP-binding proteins in vesicular transport

Recent recognition of the abundance of small GTP-binding proteins in eukaryotic cells has sparked off a search for the possible function of these proteins. Evidence is accumulating that SAR1, ARF, SEC4 and YPT1 in yeast and the rab and arf family in mammalian cells play a central role in the regulation of vesicle transport and organelle function.     

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.     

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.     

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.     

Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro.

Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue. Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK[S-farnesyl-Cys]VIM. One of these is the vacuolar protease carboxypeptidase Y. Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity. These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors. They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2. The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline. We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase. The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides. Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.     

SAM: a novel motif in yeast sterile and Drosophila polyhomeotic proteins.

Single copies of an approximately 65-70 residue domain are shown to be present in the sequences of 14 eukaryotic proteins, including yeast byr2, STE11, ste4, and STE50, which are essential participants in sexual differentiation. This domain, named SAM (sterile alpha motif), appears to participate in other developmental processes because it is also present in Drosophila polyhomeotic gene product and related homologues, which are thought to regulate determination of segmental specification in early embryogenesis. Its appearance in byr2 and STE11, which are MEK kinases, and in proteins containing pleckstrain homology, src homology 3, and discs-large homologous region domains, suggests possible participation in signal transduction pathways.     

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.     

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.     

Modularity of the slit protein. Characterization of a conserved carboxy-terminal sequence in secreted proteins and a motif implicated in extracellular protein interactions.

Since our characterization of the slit cDNA sequence, encoding a protein secreted by glial cells and involved in the formation of axonal pathways in Drosophila, we have discovered that the protein contains two additional sequence motifs that are highly conserved in a variety of proteins. A search of the GenPept database with the 73 amino acids at the carboxy terminus of slit revealed that this region contains significant similarity to a carboxy-terminal domain found in six other exported proteins. This observation has allowed us to define a new carboxy-terminal protein motif. In addition, comparisons with a 202 amino acid domain residing between epidermal growth factor (EGF) repeats in slit shows this region to be conserved in laminin, agrin and perlecan and, strikingly, also to lie between EGF repeats in both agrin and perlecan. Our analysis suggests this motif is involved in mediating interactions among extracellular proteins. Consistent with our previous characterization of the slit protein, both new motifs are found only in extracellular proteins. The identification of these two conserved motifs in slit reveals that the entire 1469 amino acids of the protein are made up of modular regions similar to those conserved in other extracellular proteins.     

The novel EPTP repeat defines a superfamily of proteins implicated in epileptic disorders

Recent studies suggest that mutations in the LGI1/Epitempin gene cause autosomal dominant lateral temporal epilepsy. This gene encodes a protein of unknown function, which we postulate is secreted. The LGI1 protein has leucine-rich repeats in the N-terminal sequence and a tandem repeat (which we named EPTP) in its C-terminal region. A redefinition of the C-terminal repeat and the application of sensitive sequence analysis methods enabled us to define a new superfamily of proteins carrying varying numbers of the novel EPTP repeats in combination with various extracellular domains. Genes encoding proteins of this family are located in genomic regions associated with epilepsy and other neurological disorders.     

The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins.

Ras-related small GTP-binding proteins (SMGs) exist in a cytosolic and a membrane-bound pool. The mechanism regulating the intracellular distribution of SMGs remains to be elucidated. We have, therefore, investigated the properties of SMGs expressed in cells of the insulin-secreting lines RINm5F and HIT-T15. Phase-partitioning analysis revealed that smg25A/rab3A as well as all the SMGs in the 23-27 kDa range, labeled by radioactive GTP after blotting, were hydrophobic, regardless of their subcellular distribution. In contrast, the cytosolic forms of ADP ribosylation factor, rho, and CDC42 were hydrophilic. The cytosolic pool of the 23-27-kDa group, including smg25A/rab3A, sedimented in a sucrose density gradient as complexes with an apparent M(r) of about 80,000, whereas rho and CDC42 were recovered in 45-kDa complexes. ARF, however, was uncomplexed (M(r) close to 20,000). The 80-kDa aggregates were likely to be formed by 1:1 complexes with the regulatory protein smg25/GDP dissociation inhibitor (smg25/GDI). In fact, pure smg25/GDI by sucrose gradient exhibited a molecular mass of 55 kDa, but cosedimented with the 80-kDa complexes in cytosolic extracts of insulin-secreting cells. Moreover, purified smg25/GDI was able to extract the SMGs of the 23-27-kDa group from the membranes. Similarly, in cytosolic extracts, rho/GDI cosedimented with the 45-kDa aggregates. Blocking the synthesis of isoprenoid groups with lovastatin resulted in the appearance in the cytosol of SMGs that were hydrophilic. These SMGs were found to sediment with an apparent M(r) close to 25,000 and to be unable to form complexes with smg25/GDI. Lovastatin treatment also caused the accumulation of the noncomplexed form of CDC42 but not of rho proteins. We propose that 1) except for ARF, all the SMGs detected in the cytosol of insulin-secreting cells are associated in 1:1 complexes with their regulatory proteins; 2) the different SMGs can be subdivided into functional groups according to the regulatory protein bound to them; 3) the formation of the 80-kDa complexes with smg25/GDI and of the CDC42 complexes with rho/GDI necessitate the correct carboxyl-terminal post-translational modification of the SMGs.     

Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.     

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.     

Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.     

A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif.

The homeobox, a 183 bp DNA sequence element, was originally identified as a region of sequence similarity between many Drosophila homeotic genes. The homeobox codes for a DNA-binding motif known as the homeodomain. Homeobox genes have been found in many animal species, including sea urchins, nematodes, frogs, mice and humans. To isolate homeobox-containing sequences from the plant Arabidopsis thaliana, a cDNA library was screened with a highly degenerate oligonucleotide corresponding to a conserved eight amino acid sequence from the helix-3 region of the homeodomain. Using this strategy two cDNA clones sharing homeobox-related sequences were identified. Interestingly, both of the cDNAs also contain a second element that potentially codes for a leucine zipper motif which is located immediately 3'' to the homeobox. The close proximity of these two domains suggests that the homeodomain-leucine zipper motif could, via dimerization of the leucine zippers, recognize dyad-symmetrical DNA sequences.