Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.     

Dependence of Ypt1 and Sec4 membrane attachment on Bet2

Many small GTP-binding proteins are synthesized as soluble proteins that are post-translationally modified as a prerequisite for membrane attachment. Ypt1 and Sec4 are homologous Raslike GTP-binding proteins that have been proposed to regulate the specificity of vesicular traffic at different stages of the secretory pathway by cycling on and off membranes. Here we show that BET2, initially identified as a gene required for transport from endoplasmic reticulum to Golgi apparatus in yeast, encodes a factor that is needed for the membrane attachment of Ypt1 and Sec4. DNA sequence analysis has revealed that Bet2 is homologous to Dpr1 (Ram1), an essential component of a protein prenyltransferase that modifies Ras, enabling it to attach to membranes. We propose that Bet2 modifies Ypt1 and Sec4 in an analogous manner.     

Exocytotic fusion is activated by Rab3a peptides.

Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion. We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells. Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers. Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion. We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S. The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP. This suggests that sustained activation of a Rab3 protein causes exocytotic fusion. The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.     

Hypervariable C-terminal domain of rab proteins acts as a targeting signal

Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast. Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro. In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways. To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation. Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway.     

Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway.

The transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems. Yeast genes encoding cytosolic components that participate in vesicular traffic were first identified from the collection of conditional-lethal sec-(secretory) mutants. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular membranes. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec7 protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with Sec7p-specific antibodies by immuno-isolation for biochemical and electron microscopic characterization. Sec7p on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies, implicate Sec7p as part of a (non-clathrin) vesicle coat. This Sec7p-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway.     

Yeast and mammalian ras proteins have conserved biochemical properties

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.     

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.     

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.     

Sequential interactions with Sec23 control the direction of vesicle traffic

How the directionality of vesicle traffic is achieved remains an important unanswered question in cell biology. The Sec23p/Sec24p coat complex sorts the fusion machinery (SNAREs) into vesicles as they bud from the endoplasmic reticulum (ER). Vesicle tethering to the Golgi begins when the tethering factor TRAPPI binds to Sec23p. Where the coat is released and how this event relates to membrane fusion is unknown. Here we use a yeast transport assay to demonstrate that an ER-derived vesicle retains its coat until it reaches the Golgi. A Golgi-associated kinase, Hrr25p (CK1δ orthologue), then phosphorylates the Sec23p/Sec24p complex. Coat phosphorylation and dephosphorylation are needed for vesicle fusion and budding, respectively. Additionally, we show that Sec23p interacts in a sequential manner with different binding partners, including TRAPPI and Hrr25p, to ensure the directionality of ER-Golgi traffic and prevent the back-fusion of a COPII vesicle with the ER. These events are conserved in mammalian cells.     

The Rab8 GTPase regulates apical protein localization in intestinal cells

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.     

Legionella pneumophila proteins that regulate Rab1 membrane cycling

Rab1 is a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells. The intracellular pathogen Legionella pneumophila subverts Rab1 function to create a vacuole that supports bacterial replication by a mechanism that is not well understood. Here we describe L. pneumophila proteins that control Rab1 activity directly. We show that a region in the DrrA (defect in Rab1 recruitment A) protein required for recruitment of Rab1 to membranes functions as a guanine nucleotide dissociation inhibitor displacement factor. A second region of the DrrA protein stimulated Rab1 activation by functioning as a guanine nucleotide exchange factor. The LepB protein was found to inactivate Rab1 by stimulating GTP hydrolysis, indicating that LepB has GTPase-activating protein activity that regulates removal of Rab proteins from membranes. Thus, L. pneumophila encodes proteins that regulate three distinct biochemical reactions critical for Rab GTPase membrane cycling to redirect Rab1 to the pathogen-occupied vacuole and to control Rab1 function.     

A genomic perspective on membrane compartment organization

Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.     

TRAPPI tethers COPII vesicles by binding the coat subunit Sec23

The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.     

Differential regulation of rasGAP and neurofibromatosis gene product activities

The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.     

Requirement for GTP hydrolysis in the formation of secretory vesicles

The specificity of vesicular transport in a cell is determined by the formation of vesicles with specific contents from a donor compartment and their selective fusion with the appropriate acceptor compartment. Several of the latter fusion steps have been investigated in detail using cell-free systems, and work with these systems as well as genetic evidence has revealed a role for GTP-binding proteins in membrane fusion processes. We have reconstituted the formation of constitutive secretory vesicles and immature secretory granules from the trans Golgi network in a cell-free system. We show here that the budding of both types of post-Golgi vesicles is inhibited by non-hydrolysable analogues of GTP, which suggests a more widespread role for GTP-binding proteins in membrane traffic than previously assumed.     

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Topological restriction of SNARE-dependent membrane fusion

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.     

A single GTP-binding protein regulates K+-channels coupled with dopamine, histamine and acetylcholine receptors

Recently, a GTP-binding protein sensitive to islet activating protein (IAP) has been suggested to be important in producing K+-currents when the muscarinic receptor of the atrial muscle is activated by acetylcholine (ACh). Here we confirm the blocking effects of IAP and GTP gamma S (a nonhydrolysable analogue of GTP) on the ACh-induced K+-current recorded from the ganglion cells of the sea slug Aplysia and compare their effects on histamine (HA)-induced and dopamine (DA)-induced K+-currents. Intracellular injections of IAP irreversibly and selectively block the openings of K+-channels activated by either ACh, HA, or DA without affecting the resting potential or conductance states of the membranes. Intracellular application of GTP gamma S alone caused extremely slow, irreversible opening of K+-channels; however, repetitive receptor activations significantly increase the rate of the GTP gamma S effect. These results strongly suggest that a GTP-binding protein such as Gi regulates the opening of K+-channels coupled with these receptors.     

The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.