A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.A large number of eukaryotic intracellular proteins are post-translationally modified by the covalent attachment of either 15 or 20 carbon isoprenoids known as farnesyl or geranylgeranyl, respectively. This process (referred to as protein prenylation) affects lipases, kinases, inositol and protein-tyrosine phosphatases, lamins, and most of the small GTPases (1–3). Protein prenylation was shown to enable reversible association of modified proteins with lipid bilayers and to modulate protein-protein interactions (4–6).The farnesyl group is attached to the cysteine of the C-terminal motif known as the CaaX box, where “a” indicates aliphatic amino acids and X is usually serine, methionine, glutamine, alanine, or threonine (3). Farnesyltransferase (FTase)³ consists of two subunits, the α-subunit and the β-subunit (Ram2p and Ram1p in yeast). The α-subunit is shared by the geranylgeranyl transferase (GGTase I), whereas the β-subunit is unique for FTase (7).The peroxisome biogenesis protein (peroxin) Pex19p is one of a few farnesylated non-GTPases that are conserved between yeast and humans. Pex19p was initially identified as a prenylated protein (PxF) (8, 9) or housekeeping gene product (HK33) (10). A loss-of-function mutation in human PEX19 is associated with complementation group CG-J/CG-14 of Zellweger syndrome (11). In the absence of Pex19p, cells lack functional peroxisomes (11–13). Pex19p is mostly cytosolic and interacts with all peroxisomal membrane proteins (PMPs) analyzed (14–16).Different and not all exclusive models have been proposed for Pex19p function. First, Pex19p might be an import receptor for PMPs that recognizes its substrates in the cytosol and delivers them to the peroxisomal membrane (15, 17, 18). This function would be analogous to that of the peroxisomal import receptors Pex5p and Pex7p, which recognize and deliver matrix proteins with PTS1 (peroxisomal targeting signal type 1) and PTS2 to peroxisomes (19). Second, Pex19p might act as a PMP chaperone that prevents newly synthesized PMPs from aggregation and degradation in the cytosol (17, 20). Third, Pex19p might act as a PMP membrane insertion factor (14, 16). Fourth, Pex19p might be required as an association/dissociation factor of membrane protein complexes (21) and has been reported to be required for the targeting of Pex3p from the ER to the peroxisomal membrane (22). Finally, Pex19p function is dependent on Pex3p, which serves as a docking factor at the peroxisomal membrane (12, 22–24). All models agree on the importance of PMP recognition for Pex19p function (25).Pex19p shows only a moderate degree of sequence conservation, with less than 20% amino acid identity between yeast and human Pex19p. Its CaaX box, however, has been retained throughout evolution (see Fig. 1). Information on the status and the requirement of Pex19p farnesylation has so far been available only through often conflicting side observations. Mammalian PEX19 was described to be partially farnesylated in CHO-K1 cells (11), but other studies with human fibroblasts challenged the relevance of Pex19p farnesylation (15, 26). It was speculated that in Saccharomyces cerevisiae, farnesylation is required for an essential aspect of Pex19p function (12). This notion was recently contradicted (27). Work on other yeasts similarly suggested that farnesylation would be dispensable for Pex19p function (13, 28, 29).Open in a separate windowFIGURE 1.Pex19p is completely farnesylated in vivo, independent of peroxisome induction and Pex3p. A and B, Pex19p is fully modified by yeast FTase in vivo. Whole cell lysates from non-induced cells of the indicated strains were analyzed by immunoblotting. Blots were probed with anti-Pex19p antibodies. The non-farnesylated form of Pex19p of a Δram1 mutant (arrowhead) cannot be detected in extracts from wild-type yeast (arrow) (A), whereas it reappears after reintroduction of Ram1p (B). C, the yeast farnesylation machinery can be saturated by overexpression of GST-Pex19p. A Coomassie-stained gel of purified farnesylated and non-farnesylated Pex19p is shown. GST-Pex19p was expressed under control of a copper-inducible promoter in Δpex19 and Δram1 strains and isolated by affinity chromatography. In Δram1 (right), only the non-farnesylated GST-Pex19p can be detected. In Δpex19 (left) two bands appear, corresponding to non-farnesylated GST-Pex19p (upper band) and farnesylated GST-Pex19p (lower band). D, Pex19p farnesylation levels are independent of peroxisome induction and are not affected by the absence of the Pex19p membrane anchor Pex3p. Cells were grown on YPD medium and, where indicated, washed and grown on 0.1% oleate medium for 17 h for peroxisome induction. Lysates were fractionated by centrifugation (20,000 × g, 1 h, 4 °C) and analyzed as in A. Blots were probed with antibodies against Pex19p. E, evolutionary conservation of the Pex19p farnesylation site in fungi, plant, and metazoa.In this study, we determined the in vivo farnesylation status of Pex19p and its dependence on peroxisome induction and on Pex3p. We discovered that Pex19p is fully modified by FTase and investigated whether Pex19p farnesylation is required for PMP recognition and stability. By peptide blots, two-hybrid analysis, and fluorescence polarization titration, we showed that farnesylation increases the affinity for PMPs by a factor of about 10. Last, we provide evidence that the interaction between farnesylated Pex19p and PMPs is achieved through a farnesylation-induced structural change in Pex19p rather than through direct farnesyl-PMP interaction. Our results exemplify the biological relevance of isoprenylation-dependent protein-protein interactions.     

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.     

Solution Structure of Human Pex5��Pex14��PTS1 Protein Complexes Obtained by Small Angle X-ray Scattering

The Pex5p receptor recognizes newly synthesized peroxisomal matrix proteins which have a C-terminal peroxisomal targeting signal to the peroxisome. After docking to protein complexes on the membrane, these proteins are translocated across the membrane. The docking mechanism remains unclear, as no structural data on the multicomponent docking complex are available. As the interaction of the cargo-loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step, we have investigated the solution structure of these complexes by small angle x-ray scattering and static light scattering. Titration studies yielded a 1:6 stoichiometry for the Pex5p·Pex14p complex, and low resolution structural models were reconstructed from the x-ray scattering data. The free full-length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. The model of the complex reveals that the N terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with the Pex5p moiety. These results suggest that the extended structure of Pex5p may play a role in interactions with other substrates such as lipids and membrane proteins during the formation of functional multiprotein complexes.Peroxisomes are ubiquitous organelles in eukaryotes which are involved in different metabolic pathways (1). Peroxisomal matrix proteins, which contain a peroxisomal targeting signal (PTS),⁴ are imported into the peroxisome by recognition of two different import receptors, Pex5p or Pex7p. These receptors recognize specific signal sequences, PTS1 and PTS2, respectively (1). At the molecular level the C-terminal PTS1 signal is bound in a central cavity of the ring-like structure of the seven tetrapeptide repeat (TPR) domains of the C-terminal part of Pex5p (Pex5p(C)) (2–5). It was recently proposed that some of the structural principles of the Pex5p/cargo interaction may also apply to the PTS2 cargo recognition of the Pex7p receptor (5).The next step of PTS-protein import, docking of the cargo loaded receptor to the translocon, involves the peroxisomal protein Pex14p (6). Multiple Pex14p binding sites with di-aromatic pentapeptide motifs (WXXX(F/Y)) were shown to be present in the N terminus of Pex5p (7–9). The number of these motifs, however, varies among species. The human Pex5p receptor, which has been investigated in this contribution, has a total of seven motifs. A recent NMR structure of the N-terminal domain of Pex14p and the first WXXX(F/Y) motif of Pex5p reveals an α-helical conformation of the motif (10). Interactions between Pex5p and other proteins and by their association with the peroxisomal membrane possibly lead to dissociation of the PTS-protein from Pex5p (11–13). The exact sequence of events in the import mechanism remains, however, unknown. It is in particular unclear how, in contrast with other organelles, peroxisomes can import folded oligomeric, functional proteins (14).Previous biophysical work indicated that the N terminus half of Pex5p is unfolded in vitro (15, 16). Recent protease sensitivity assays showed that the proteolytic profiles of the full-length receptor Pex5p(F) change in the presence of PTS1 peptide and the Pex13p Src homology 3 domain, which is another docking factor (16, 17), indicating conformational changes of Pex5p upon binding these receptor ligands. Furthermore, it was found that Pex5p may even traverse the peroxisomal membrane, leaving only a small N-terminal fragment in the cytosol while exposing the C-terminal TPR domain to the luminal side of the membrane (11).Although recognition of many PTS cargos seems to be confined to the C-terminal TPR domains of Pex5p, it has become clear that the N-terminal part of Pex5p is primarily involved in docking of the receptor onto the peroxisomal membrane and other docking factors. Because only poorly diffracting crystals have been purified to date, we investigated its solution structure by small angle x-ray scattering (SAXS) and static light scattering (SLS). Complexes with the PTS1 cargo sterol carrier protein 2 (SCP2), which functions as lipid transfer protein, were also studied as the crystal structure of Pex5p(C)/SCP2 is already known (4). Our results indicate that human Pex5p(F) is a monomer with an extended N terminus. The stoichiometry of Pex5p(F)·Pex14p(N)·PTS1 complex has been assessed by titration with SAXS, SLS, and gel filtration, and a low resolution structural model of the complex has been reconstructed in which Pex5p(F) remains extended upon Pex14p(N) binding.     

Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.Cytoskeletal elements have previously been demonstrated to be involved in several aspects of virus assembly (39, 66). For example, vaccinia virus has been shown to associate with actin during its release from the plasma membrane (15), while adenovirus is transported through the cytoplasm to the nucleus through its interaction with microtubules (17, 38). Actin has been implicated in the transport of baculovirus nucleocapsids to the nucleus (10). Other viruses contain actin in their envelopes along with viral surface glycoproteins, implying some role in the budding process (34, 54, 58). In addition, cytochalasin D, a disruptor of microfilaments, has been shown to impair the assembly of a number of different viruses (18, 42, 45). Most viruses use preexisting microtubule or microfilament proteins derived from host cells in these processes. However, we have recently demonstrated that insect poxviruses establish their own filament network during the later stages of infection, using a protein encoded by the viral genome (2).Entomopoxviruses (EPVs) are insect pathogens which replicate in the cytoplasm of infected cells and are members of the poxvirus family (reviewed in references 3 to 5 and 22). The genomes of these viruses consist of linear double-stranded DNA molecules which are 130 to 300 kb in length. Amsacta moorei EPV (AmEPV) can be grown in cultured insect cells and is the most studied member of this group of viruses (22–25, 27, 40, 50). AmEPV derives its name from the Indian red army worm, a larva from the Lepidoptera family and the host from which the virus was originally isolated (23, 25, 50). Baculoviruses also infect Lepidoptera larvae but instead replicate in the nuclei of their host cells (44). A number of baculoviruses have been studied, but knowledge of Autographa californica nuclear polyhedrosis virus (AcNPV), which infects a wide variety of larvae including that of the alfalfa leaf hopper, is most extensive (44). This virus is used routinely to produce recombinant proteins in insect virus expression systems (36, 44, 46, 49).A common property of EPVs and baculoviruses is the formation of large intracellular structures known as occlusion bodies which assemble during the late stages of viral infection. Virions are embedded within these occlusion bodies, and the process serves to protect the virus from the external environment. In the case of baculoviruses, the occlusion bodies are called polyhedra and are composed predominantly of a 31-kDa protein called polyhedrin (52). The occlusion bodies of EPVs are known as spheroids and consist mainly of a 110-kDa protein known as spheroidin (6, 9, 27, 55). Spheroidin and polyhedrin do not appear to exhibit sequence homology (6, 27, 52). A multilamellar envelope also appears to surround both polyhedra and spheroids and may help to stabilize these structures during assembly (2, 53).During the late phases of AmEPV and baculovirus infections, large bundles of filaments also appear to accumulate in the infected insect cells. In the case of AmEPV, these structures are present in the cytoplasm (2, 22, 23, 40), while those found in cells infected with baculoviruses reside both in the cytoplasm and in the nucleus (1, 14, 57). Baculovirus fibrils are composed primarily of a 10-kDa protein called p10 (47, 59). The p10 gene sequences from AcNPV, Orgyia pseudotsugata nuclear polyhedrosis virus (OpNPV), Bombyx mori nuclear polyhedrosis virus, Perina nuda nuclear polyhedrosis virus, Spodoptera exigua nuclear polyhedrosis virus (SeNPV), and Choristoneura fumiferana nuclear polyhedrosis virus (CfNPV) have been reported (13, 32, 35, 66–68). Although the different p10 protein sequences only exhibit 39 to 51% identity and molecules from different species cannot interact with one another, it is believed that the polypeptides must be structurally and functionally similar (61, 66). Deletion mutagenesis of AcNPV p10 has demonstrated that both the amino- and carboxy-terminal regions of this protein are necessary for the formation of filaments in the infected cell (60). Other studies have assigned an aggregation function to the amino-terminal half of p10 (63, 65), and it has been shown that this region contains a coiled-coil domain which is conserved among the different baculoviruses (66). It is tempting to speculate that p10 aggregation is the result of coiled-coil interaction, but direct evidence for this hypothesis is lacking. The precise role of the carboxy terminus of p10 is still unclear, although it has been proposed to interact with tubulin (11). Deletion of the entire p10 open reading frame (ORF) through homologous recombination produces a mutant virus which is still capable of replication both in vitro and in vivo but produces fragile polyhedra with fragmented polyhedral envelopes (26, 64, 65). The p10 protein has also been implicated in disintegration of the nuclear envelope of the host cell, and this function appears to be associated with the carboxy terminus of this protein (61, 65).Our laboratory (2) recently demonstrated that the cytoplasmic filaments, which characterize the late stages of infection by AmEPV, are composed primarily of a 156-amino-acid protein called FALPE (filament-associated late protein of EPVs). These filaments are closely associated with the spheroids and their membrane envelopes. FALPE is a phosphoprotein which migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 25/27-kDa doublet. This protein also contains an unusual proline-glutamic acid repeat region spanning 20 residues in the carboxy terminus of the polypeptide. The ultrastructure and close association of this protein with the occlusion bodies of AmEPV suggested that FALPE and p10 played analogous roles during infections by the respective viruses.This article addresses the structural and functional similarities between FALPE and p10. These two viral proteins are known to be major components of filamentous structures, but it is not known whether additional viral or cellular proteins cooperate during the polymerization process. In this report, we provide insight into the mechanisms which produce filaments in cells infected with either baculoviruses or EPVs. We demonstrate that p10 and FALPE can produce filaments in the absence of other viral gene products. Using the yeast two-hybrid system and a chemical cross-linking agent, we obtained evidence for self-association of either FALPE or p10. Finally, the polypeptide regions of FALPE and p10 which are required for self-association and subsequent filament formation are mapped.     

Sec35p,a Novel Peripheral Membrane Protein,Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components (Rothman, 1994). The process can be divided into three stages: (a) vesicle budding, (b) vesicle docking, and (c) membrane fusion, with distinct sets of proteins mediating each phase. The budding step involves recruitment of coat proteins to the membrane and culminates with the release of coated vesicles (Schekman and Orci, 1996). The docking reaction is likely to require a set of integral membrane proteins on the vesicle and target membranes, termed v-SNAREs¹ and t-SNAREs (vesicle- and target membrane-soluble N-ethylmaleimide–sensitive fusion protein [NSF] attachment protein [SNAP] receptors, respectively), that are thought to confer specificity through their pair-wise interactions (Söllner et al., 1993b ). Small GTP-binding proteins of the rab family also assist in the docking process (Ferro-Novick and Novick, 1993), but their precise function is not known. The fusion step ensues after docking and results in the delivery of the vesicular cargo to the next compartment in the secretory pathway.Vesicular transport from the ER to the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively characterized. Transport vesicle budding involves the assembly of the COPII coat, composed of the Sec13p/Sec31p (Pryer et al., 1993; Salama et al., 1993; Barlowe et al., 1994) and Sec23p/Sec24p heterodimers (Hicke and Schekman, 1989; Hicke et al., 1992), under the direction of an integral membrane protein, Sec12p (Nakano et al., 1988; Barlowe and Schekman, 1993), a small GTPase, Sar1p (Nakano and Muramatsu, 1989), and a multidomain protein, Sec16p (Espenshade et al., 1995; Shaywitz et al., 1997). Docking is thought to require a tethering event mediated by Uso1p (Cao et al., 1998), the yeast homologue of mammalian p115 (Barroso et al., 1995; Sapperstein et al., 1995), followed by or concurrent with the interaction of a set of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p (Newman and Ferro-Novick, 1987; Newman et al., 1990; Ossig et al., 1991; Shim et al., 1991; Søgaard et al., 1994) and perhaps Ykt6p (Søgaard et al., 1994; McNew et al., 1997), with the cognate t-SNARE on the Golgi, Sed5p (Hardwick and Pelham, 1992). For some time it was thought that fusion may be initiated by disassembly of the v/t-SNARE complex (Söllner et al., 1993a ) by yeast SNAP, Sec17p, (Griff et al., 1992) and NSF, Sec18p (Eakle et al., 1988; Wilson et al., 1989). However, this concept has been challenged by studies with a yeast system that reconstitutes homotypic vacuolar fusion, which suggests the action of Sec18p is before vesicle docking (Mayer et al., 1996; Mayer and Wickner, 1997). In addition, a prefusion role for NSF has been supported by the recent finding that liposomes bearing SNAREs alone can fuse in the absence of NSF (Weber et al., 1998).Several proteins involved in the regulation of yeast ER to Golgi v/t-SNARE complex assembly have been identified, including Ypt1p, Uso1p, and Sly1p. Ypt1p is a member of the rab family of small GTP-binding proteins that have been identified as important components of almost every stage in the secretory pathway (Ferro-Novick and Novick, 1993). Hydrolysis of GTP by rab-like proteins has been hypothesized to provide the regulatory switch that controls the fidelity of vesicular transport (Bourne, 1988). A second protein, Uso1p (Nakajima et al., 1991), appears to function in the same pathway as Ypt1p (Sapperstein et al., 1996), and both proteins have been demonstrated to be essential for SNARE complex assembly (Søgaard et al., 1994; Sapperstein et al., 1996; Lupashin and Waters, 1997). The third protein, Sly1p, is associated with the t-SNARE Sed5p (Søgaard et al., 1994). SLY1 is an essential gene in yeast (Dascher et al., 1991; Ossig et al., 1991), and Sly1p is required for ER to Golgi transport in vitro (Lupashin et al., 1996) and in vivo (Ossig et al., 1991). However, several lines of evidence, particularly from Sly1p homologues in other organisms, indicate that Sly1p may also function as a negative regulator of v/t-SNARE complex assembly, perhaps by preventing the association of the v- and t-SNAREs (Hosono et al., 1992; Pevsner et al., 1994; Schulze et al., 1994). A dominant allele of SLY1, termed SLY1-20, is capable of suppressing mutations in YPT1 and USO1, including complete deletions (Dascher et al., 1991; Sapperstein et al., 1996). Thus, in the presence of Sly1-20p, two components required for SNARE complex assembly are no longer essential. We have proposed a model (Sapperstein et al., 1996; Lupashin and Waters, 1997) in which Ypt1p and Uso1p function to relieve the inhibitory action of Sly1p on SNARE complex assembly. In this model Sly1-20p can be thought of as a noninhibitory form of SLY1 that renders Ypt1p and Uso1p superfluous.We believe that the ability of SLY1-20 to suppress defects in upstream docking regulators can be used to identify additional components involved in the regulation of vesicular docking. We have undertaken a genetic screen (to be presented elsewhere) to isolate novel components in this pathway which, when mutated, depend upon Sly1-20p for viability. In the course of this work, we discovered that two recently identified mutants, sec34 and sec35, can be suppressed by SLY1-20 and thus satisfy the criterion of our screen. These mutants were isolated in a novel screen to identify components involved in transport at any step between the ER and the trans-Golgi network (i.e., the Kex2p compartment) in yeast (Wuestehube et al., 1996). Both sec34 and sec35 accumulate the core-glycosylated form of secretory proteins at the nonpermissive temperature, indicating a block in ER to Golgi transport. Furthermore, electron microscopy indicated that both sec34 and sec35 accumulate numerous vesicles upon shift to the restrictive temperature (Wuestehube et al., 1996), a hallmark of genes whose protein products are involved in the docking or fusion phase of transport (Kaiser and Schekman, 1990). In this report we describe the cloning of SEC35 and analysis of its genetic interactions with other secretory genes. Strong genetic interaction between SEC35 and SLY1, YPT1, and USO1 suggests that Sec35p may function in vesicle docking. To test this possibility, we devised an in vitro transport assay that depends on the addition of purified Sec35p and Uso1p. Vesicles synthesized in the absence of functional Sec35p do not fuse with the Golgi compartment and remain as freely diffusible intermediates. Upon addition of Sec35p and Uso1p, vesicles dock to the Golgi and proceed to membrane fusion. Requirements for Sec35p at the vesicle docking step correlates our genetic experiments with the biochemically distinguishable steps of vesicle docking and membrane fusion.