The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction,Molecular Transport,and Callose Biosynthesis

The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.The plasma membrane (PM)¹ is considered as one of the most interactive and dynamic supramolecular structures of the cell (1, 2). It forms a physical interface between the cytoplasm and the extracellular environment and is involved in many biological processes such as metabolite and ion transport, gaseous exchanges, endocytosis, cell differentiation and proliferation, defense against pathogens, etc. (3). Various combinations of biochemical and analytical approaches have been used to characterize the PM proteome in different organisms such as yeast, plants, and animals (4–8). Typically, PM proteins are either embedded in the phospholipid bilayer through transmembrane helices or less tightly bound to the membrane through reversible or irreversible surface interactions. In eukaryotic cells, some PM proteins are enriched in lateral lipid patches that form microdomains within the membrane (9, 10). These microdomains are considered to act as functional units that support and regulate specific biological processes associated with the PM (9, 10). Often referred to as “membrane (lipid) rafts” in animals and other organisms, they are typically described as being enriched in sphingolipids, sterols, and phospholipids that contain essentially saturated fatty acids (9–11). Early work on PM microdomains has suggested that their specific lipid composition confers resistance to certain concentrations of nonionic detergents, such as Triton X-100 and Nonidet P-40 (10, 11). Although this property has been exploited experimentally to isolate so-called detergent-resistant microdomains (DRMs), the relationship between DRMs and membrane rafts remains controversial (12). Indeed, the relation between the two is much debated, essentially because the use of Triton X-100 at 4 °C to prepare DRMs has been proposed to potentially induce the artificial formation of detergent-resistant structures whose composition may not fully reflect that of physiological membrane rafts (12). Nonetheless, DRM preparations represent an excellent system for the isolation and identification of groups of proteins—eventually associated in complexes—that tend to naturally interact with specific sets of lipids, thereby forming specialized functional units. Their biochemical characterization is therefore most useful in attempts to better understand the mode of interaction of specific proteins with sterols and sphingolipids and to gain insight into the protein composition and biological activity of subdomains from the PM.Plant DRMs have been understudied relative to their animal counterparts. Indeed, proteomic studies have been undertaken on DRM preparations from only a limited number of plant species. These include tobacco (13–15), Arabidopsis (16), barrel clover (Medicago truncatula) (17), rice (18), oat, and rye (19). These studies, essentially based on qualitative or semi-quantitative proteomics, led to the identification of hundreds of proteins involved in a large range of mechanisms, functions, and biochemical activities (15–19). Depending on the report considered, a variable proportion of the identified proteins can be intuitively linked to DRMs and potentially to PM microdomains. However, many proteins that are clearly not related to the PM and its microdomains co-purify with DRM. These include, for instance, soluble proteins from cytoplasmic metabolic pathways; histones; and ribosomal, chloroplastic, and mitochondrial proteins (15–19). Thus, there is a need to obtain a more restricted list of proteins that are specifically enriched in DRMs and that define specialized functional structures. One way to tackle this problem is through quantitative proteomics, eventually in combination with complementary biochemical approaches. Although quantitative techniques have been increasingly applied to the proteomic analysis of complex mixtures of soluble proteins, their exploitation for the characterization of membrane samples remains challenging. As a result, very few studies of plant DRMs have been based on truly quantitative methods. For instance, stable isotope labeling combined with the selective disruption of sterol-rich membrane domains by methylcyclodextrin was performed in Arabidopsis cell cultures (20). A similar approach was used to study compositional changes of tobacco DRMs upon cell treatment with the signaling elicitor cryptogenin (21). In another study, 64 Arabidopsis proteins were shown to be significantly enriched in DRMs in response to a pathogen-associated molecular pattern protein (22). Together, these few quantitative proteomics analyses suggest a role of plant membrane microdomains in signal transduction, as in mammalian cells.Although several reports describe the partial characterization of DRMs from higher plants (13–23), there are no data available to date on the protein composition of DRMs from a tree species. We have therefore employed a quantitative proteomic approach for the characterization of DRMs from cell suspension cultures of Populus trichocarpa. In addition, earlier work in our laboratory based on biochemical activity assays revealed the presence of cell wall polysaccharide synthases in DRMs from poplar (23), which suggests the existence of DRM populations specialized in cell wall biosynthesis. This concept was further supported by similar investigations performed on DRMs isolated from the oomycete Saprolegnia monoica (24). The comprehensive quantitative proteomic analysis performed here revealed enrichment in the poplar DRMs of specific carbohydrate synthases involved in callose polymerization. Consistent with the role of callose in plant defense mechanisms, additional proteins related to stress responses and signal transduction were found to be specifically enriched in the poplar DRMs, together with proteins involved in molecular transport. To date, our report is the only analysis available of the DRM proteome of a tree species based on quantitative proteomics. The specific biochemical properties of the 80 proteins significantly enriched in DRMs are described and examined in relation to their localization in membrane microdomains. The relationship between poplar DRMs and molecular transport, signal transduction, stress responses, and callose biosynthesis is discussed, with support from a hypothetical model that integrates the corresponding enriched proteins.     

ERS-24, a Mammalian v-SNARE Implicated in Vesicle Traffic between the ER and the Golgi

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.Newly formed transport vesicles have to be selectively targeted to their correct destinations, implying the existence of a set of compartment-specific proteins acting as unique receptor–ligand pairs. Such proteins have now been identified (Söllner et al., 1993a ; Rothman, 1994): one partner efficiently packaged into vesicles, termed a v-SNARE,¹ and the other mainly localized to the target compartment, a t-SNARE. Cognate pairs of v- and t-SNAREs, capable of binding each other specifically, have been identified for the ER–Golgi transport step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994), the Golgi–plasma membrane transport step (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994) in Saccharomyces cerevisiae, and regulated exocytosis in neuronal synapses (Söllner et al., 1993a ; for reviews see Scheller, 1995; Südhof, 1995). Additional components, like p115, rab proteins, and sec1 proteins, appear to regulate vesicle docking by controlling the assembly of SNARE complexes (Søgaard et al., 1994; Lian et al., 1994; Sapperstein et al., 1996; Hata et al., 1993; Pevsner et al., 1994).In contrast with vesicle docking, which requires compartment-specific components, the fusion of the two lipid bilayers uses a more general machinery derived, at least in part, from the cytosol (Rothman, 1994), which includes an ATPase, the N-ethylmaleimide–sensitive fusion protein (NSF) (Block et al., 1988; Malhotra et al., 1988), and soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Clary and Rothman, 1990; Whiteheart et al., 1993). Only the assembled v–t-SNARE complex provides high affinity sites for the consecutive binding of three SNAPs (Söllner et al., 1993b ; Hayashi et al., 1995) and NSF. When NSF is inactivated in vivo, v–t-SNARE complexes accumulate, confirming that NSF is needed for fusion after stable docking (Søgaard et al., 1994).The complex of SNAREs, SNAPs, and NSF can be isolated from detergent extracts of cellular membranes in the presence of ATPγS, or in the presence of ATP but in the absence of Mg²⁺, and sediments at ∼20 Svedberg (20S particle) (Wilson et al., 1992). In the presence of MgATP, the ATPase of NSF disassembles the v–t-SNARE complex and also releases SNAPs. It seems likely that this step somehow initiates fusion.To better understand vesicle flow patterns within cells, it is clearly of interest to identify new SNARE proteins. Presently, the most complete inventory is in yeast, but immunolocalization is difficult in yeast compared with animal cells, and many steps in protein transport have been reconstituted in animal extracts (Rothman, 1992) that have not yet been developed in yeast. Therefore, it is important to create an inventory of SNARE proteins in animal cells. The most unambiguous and direct method for isolating new SNAREs is to exploit their ability to assemble together with SNAPs and NSF into 20S particles and to disassemble into subunits when NSF hydrolyzes ATP. Similar approaches have already been successfully used to isolate new SNAREs implicated in ER to Golgi (Søgaard et al., 1994) and intra-Golgi transport (Nagahama et al., 1996), in addition to the original discovery of SNAREs in the context of neurotransmission (Söllner et al., 1993a ).Using this method, we now report the isolation and detailed characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE that is localized to the ER and Golgi. ERS-24 is found in transport vesicles associated with the transitional areas of the ER and with the rims of Golgi cisternae, suggesting a role for ERS-24 in vesicular transport between these two compartments.     

Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.Microtubules (MTs)¹ in vertebrate somatic cells are involved in intracellular transport and distribution of membranous organelles. Fundamental to this role are their tightly controlled, polarized organization, and unusual dynamic properties (Hirokawa, 1994) and their interaction with a complex set of MT-based motor proteins (Hirokawa, 1996; Sheetz, 1996; Goodson et al., 1997). During mitosis, they contribute to the motility of centrosomes, the construction of spindle poles (Karsenti et al., 1996; Merdes and Cleveland, 1997), and the dynamic movements of kinetochores (Rieder and Salmon, 1994) and chromosome arms (Barton and Goldstein, 1996; Vernos and Karsenti, 1996). The motor protein cytoplasmic dynein, drives the transport toward MT minus-ends of a variety of subcellular organelles (Schnapp and Reese, 1989; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynactin is a molecular complex originally identified as being essential for dynein-mediated movement of salt-washed vesicles in vitro (reviewed in Schroer, 1996; Schroer and Sheetz, 1991). Genetic studies in fungi, yeast, and flies have shown that the two complexes function together to drive nuclear migration, spindle and nuclear positioning and to permit proper neuronal development (Eshel et al., 1993; Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; McGrail et al., 1995; Karsenti et al., 1996). Biochemical studies suggest a direct interaction between certain subunits of dynein and dynactin (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). In vivo, the two molecules may bind one another transiently, since they have not been isolated as a stable complex.There is good evidence indicating that the dynein/dynactin complex, together with other motors (Eg5, and a minus-end oriented kinesin-related protein) and a structural protein (NuMa), drive the focusing of free microtubule ends into mitotic spindle poles (Merdes and Cleveland, 1997; Waters and Salmon, 1997)_. A trimolecular complex composed of NuMa and dynein/dynactin may be crucial in this process in both acentriolar (Merdes et al., 1996), and centriolar spindles (Gaglio et al., 1997). A number of findings also indicate that the combined actions of dynein and dynactin at the kinetochore contribute to chromosome alignment in vertebrate somatic cells. First, the initial interaction between polar spindle MTs and kinetochores seems to involve a tangential capture event (Merdes and De Mey, 1990; Rieder and Alexander, 1990) which is followed by a poleward gliding along the surface lattice of the MT (Hayden et al., 1990). Both in vivo and in vitro (Hyman and Mitchison, 1991) this gliding movement appears similar to the dynein-mediated retrograde transport of vesicular organelles along MTs. Consistent with this is the finding that both dynein (Pfarr et al., 1990; Steuer et al., 1990) and its activator, dynactin (Echeverri et al., 1996), are present at prometaphase kinetochores. Overexpression of dynamitin, a 50-kD subunit of the dynactin complex, results in the partial disruption of the dynactin complex and in the loss, from kinetochores, of dynein, as well as dynactin. Therefore, it has been proposed that dynactin mediates the association of dynein with kinetochores. Abnormal spindles with poorly focused poles are observed and the cells become arrested in pseudoprometaphase (Echeverri et al., 1996). Despite these findings, rigorous proof for a role of the dynein motor complex in kinetochore motility is still lacking, and its role may differ between lower and higher eucaryotes, and between mitosis and meiosis.CLIP-170 (Rickard and Kreis, 1996) is needed for in vitro binding of endocytic transport vesicles to MTs (Pierre et al., 1992). It is a nonmotor MT-binding protein that accumulates preferentially in the vicinity of MT plus ends and on early endosomes and endocytic transport vesicles in nondividing cells (Rickard and Kreis, 1990; Pierre et al., 1992). Like many MT-binding proteins, CLIP-170 is a homodimer whose NH₂-terminal head domains and COOH-terminal tail domains flank a central α-helical coiled-coil domain. The binding of CLIP-170 to MTs involves a 57–amino acid sequence present twice in the head domain (Pierre et al., 1992) and is regulated by phosphorylation (Rickard and Kreis, 1991). The COOH-terminal domain has been proposed to participate in targeting to endocytic membranes (Pierre et al., 1994). The fact that the latter move predominantly toward microtubule minus ends in a process most likely mediated by cytoplasmic dynein and dynactin (Aniento and Gruenberg, 1995), suggests that CLIP-170 may act in concert with this motor complex, and may be subject to regulated interactions with one or more dynactin or dynein subunits at the vesicle membrane.Here we report that during mitosis, CLIP-170 codistributes with dynein and dynactin at kinetochores, but not spindle poles. Evidence is presented that the COOH-terminal domain of CLIP-170 is responsible for its kinetochore targeting, and that this may be mediated by the complex of dynein and dynactin. The effects on mitotic progression of overexpression of wild type and several deletion mutants of CLIP-170 provide evidence for the involvement of CLIP-170 in kinetochore function early in mitosis. We also present in vivo evidence that neither CLIP-170 nor the complex of dynein and dynactin are required for formation of kinetochore fibers.     

Vid24p,a Novel Protein Localized to the Fructose-1,6-bisphosphatase–containing Vesicles,Regulates Targeting of Fructose-1,6-bisphosphatase from the Vesicles to the Vacuole for Degradation

Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.Protein degradation is an important process that is tightly regulated. In mammalian cells, serum starvation induces protein degradation by lysosomes (Dice, 1990; Hayes and Dice, 1996). Cytosolic proteins containing a pentapeptide sequence are targeted to the lysosome for degradation in a process mediated by a heat shock protein (Chiang and Dice, 1988; Chiang et al., 1989; Terlecky et al., 1992; Terlecky and Dice, 1993; Cuervo et al., 1994). The receptor protein for this selective proteolysis pathway has been identified recently to be LGP96 (Cuervo and Dice, 1996). Overexpression of the receptor protein increases the degradation of cytosolic proteins in lysosomes both in vivo and in vitro (Cuervo and Dice, 1996).In Saccharomyces cerevisiae, the vacuole is functionally homologous to the lysosome and takes up proteins by several mechanisms. Most vacuole resident proteinases such as carboxypeptidase Y (CPY)¹ enter the vacuole through the secretory pathway (Hasilik and Tanner, 1978; Hemmings et al., 1981; Rothman and Stevens, 1986; Banta et al., 1988; Jones, 1991). CPY is synthesized and processed sequentially in the ER and the Golgi. Sorting occurs in the late Golgi by the CPY receptor encoded by the PEP1/ VPS10 gene (Marcusson et al., 1994; Cooper and Stevens, 1996). CPY is delivered to the vacuole from the prevacuolar or endosomal compartment and the receptor protein recycles back to the Golgi (Marcusson et al., 1994; Cooper and Stevens, 1996). Other vacuolar proteins such as α-mannosidase or aminopeptidase I are imported from the cytosol to the vacuole, independent of the secretory pathway (Yoshihisa and Anraku, 1990; Klionsky et al., 1992; Harding et al., 1995, 1996; Scott et al., 1996). Plasma membrane proteins can be internalized by endocytosis and transported through early endosomes to late endosomes, from which they are directed to the vacuole for degradation (Davis et al., 1993; Raths et al., 1993; Kolling and Hollenberg, 1994; Schandel and Jennes, 1994; Lai et al., 1995; Riballo et al., 1995). Organelles such as peroxisomes or mitochondria can be engulfed by the vacuoles by autophagy (Takeshige et al., 1992; Tuttle and Dunn, 1995; Chiang et al., 1996). The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae cells are grown in medium containing poor carbon sources. When cells are transferred to medium containing fresh glucose, FBPase is rapidly inactivated (Gancedo, 1971). Using isogenic strains differing only at the PEP4 gene, we have demonstrated that FBPase is targeted from the cytosol to the vacuole for degradation when cells are transferred from poor carbon sources to fresh glucose (Chiang and Schekman, 1991). The PEP4 gene encodes proteinase A, whose activity is required for the maturation of proteinase B and proteinase C (Zubenko and Jones, 1981; Jones, 1991). As a result, the pep4 strain reduces the vacuolar proteolytic activity to 30% of the wild-type level (Zubenko and Jones, 1981; Jones, 1991; Chiang et al., 1996). The glucose-induced distribution of FBPase from the cytosol to the vacuole has been observed in the pep4 cell by cell fractionation techniques, immunofluorescence microscopy, and immunoelectron microscopy (Chiang and Schekman, 1991; Chiang et al., 1996). FBPase targeting into the vacuole always occurs, regardless of whether cells are transferred to glucose from acetate, ethanol, galactose, or oleate (Chiang and Schekman, 1994; Chiang et al., 1996).To dissect the FBPase degradation pathway, we have taken a genetic approach. Several vid (vacuolar import and degradation) mutants that fail to degrade FBPase in response to glucose have been isolated (Hoffman and Chiang, 1996). Most vid mutants block FBPase in the cytosol. However, in the vid14-1, vid15-1, and vid16-1 mutants, FBPase is found in punctate structures in the cytoplasm. When cell extracts from one of these mutants are fractionated, a substantial amount of FBPase is found in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these mutants (Hoffman and Chiang, 1996). This association is also observed in wild-type cells (Huang and Chiang, 1997).The FBPase-containing vesicles have been purified from wild-type cells to near homogeneity using a combination of differential centrifugation, gel filtration, and equilibrium centrifugation in sucrose gradients (Huang and Chiang, 1997). The purified fractions contain 30–40-nm-diam vesicles and are essentially free of other organelles. Kinetic studies indicate that FBPase association with these vesicles is induced by glucose, occurs only transiently, and precedes the association with the vacuole. The FBPase-containing vesicles are distinct from mitochondria, peroxisomes, endosomes, vacuoles, ER, Golgi, or transport vesicles such as the coat protein (COPI or COPII)-containing vesicles as analyzed by protein markers and electron microscopy (Huang and Chiang, 1997).The vesicles were predicted to contain proteins involved in FBPase targeting and sequestration into the vesicles, as well as proteins participating in carrying FBPase from the vesicles to the vacuole for degradation. To identify such factors, we cloned our first VID gene. The VID24 gene was identified by complementation of the degradation defect of the vid24-1 mutant. Vid24p is a novel 41-kD protein and is synthesized in response to glucose. A significant portion of the Vid24p is localized to the FBPase-containing vesicles as a peripheral protein. The deletion of Vid24p abolishes the degradation of FBPase, but does not cause significant change in growth, sporulation, germination, osmolarity sensitivity, or processing of CPY. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole. FBPase is sequestered inside the vesicles in the vid24-1 mutant, suggesting that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that is localized to the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.     

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.     

C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle.The serine/threonine kinase NIMA of Aspergillus nidulans is considered the founding member of a family of protein kinases with a possible role in cell cycle regulation (for reviews see Fry and Nigg, 1995; Lu and Hunter, 1995a ; Osmani and Ye, 1996). In A. nidulans, NIMA clearly cooperates with the Cdc2 protein kinase to promote progression into mitosis (Osmani et al., 1991), and overexpression of NIMA in a variety of heterologous species promotes a premature onset of chromosome condensation (O''Connell et al., 1994; Lu and Hunter, 1995b ). This has been interpreted to suggest evolutionary conservation of a pathway involving NIMA-related kinases (for review see Lu and Hunter, 1995a ). Indeed, kinases structurally related to NIMA are present in many species (Fry and Nigg, 1997). However, the only bona fide functional homologue of NIMA so far isolated stems from another filamentous fungus, Neurospora crassa (Pu et al., 1995), and the functional relationship between vertebrate NIMA-related kinases and fungal NIMA remains uncertain.The closest known mammalian relative to NIMA is a kinase termed Nek2 (for NIMA-related kinase 2)¹ (Fry and Nigg, 1997). This kinase undergoes cell cycle–dependent changes in abundance and activity, reminiscent of NIMA (Schultz et al., 1994; Fry et al., 1995). It is highly expressed in male germ cells (Rhee and Wolgemuth, 1997; Tanaka et al., 1997), and data have been reported consistent with a role for Nek2 in meiotic chromosome condensation (Rhee and Wolgemuth, 1997). However, overexpression of active Nek2 in somatic cells has no obvious effect on chromosome condensation; instead, it induces striking alterations in the structure of the centrosome, the principal microtubule-organizing center of mammalian cells (Fry et al., 1998). Furthermore, immunofluorescence microscopy and subcellular fractionation concur to demonstrate that endogenous Nek2 associates with centrosomes, strongly suggesting that one physiological function of this kinase may relate to the centrosome cycle (Fry et al., 1998).The mammalian centrosome is an organelle of about 1 μm in diameter. It comprises two barrel-shaped centrioles that are made of nine short triplet microtubules and are surrounded by an amorphous matrix known as the pericentriolar material (PCM) (for review see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Kimble and Kuriyama, 1992; Kalt and Schliwa, 1993; Kellogg et al., 1994; Lange and Gull, 1996). Major progress has recently been made with the demonstration that microtubules are nucleated from γ-tubulin–containing ring complexes (γ-TuRCs), which are concentrated within the PCM (Moritz et al., 1995; Zheng et al., 1995). γ-Tubulin forms complexes with Spc97/98, two evolutionarily conserved proteins first identified in budding yeast spindle pole bodies (Geissler et al., 1996; Knop et al., 1997; Stearns and Winey, 1997), and there is also evidence for an important role of pericentrin and other coiled-coil proteins in organizing γ-TuRCs into higher order lattice structures (Doxsey et al., 1994; Dictenberg et al., 1998). However, in spite of this recent progress, it is clear that the inventory of centrosome components is far from complete.Centrosome structure and function is regulated in a cell cycle–dependent manner (for reviews see Mazia, 1987; Kellogg et al., 1994; Tournier and Bornens, 1994). Once in every cell cycle, and beginning around the G1/S transition, centrioles are duplicated (e.g., Kuriyama and Borisy, 1981a ; Vorobjev and Chentsov, 1982; Kochanski and Borisy, 1990; Chrétien et al., 1997). Late in G2, centrosomes then grow in size (a process referred to as maturation) through the recruitment of additional PCM proteins (Rieder and Borisy, 1982; Kalt and Schliwa, 1993; Lange and Gull, 1995). At the G2/M transition, the duplicated centrosomes separate and migrate to opposite ends of the nucleus. Concomitantly, their microtubule-nucleating activities increase dramatically in preparation for spindle formation (McGill and Brinkley, 1975; Snyder and McIntosh, 1975; Gould and Borisy, 1977; Kuriyama and Borisy, 1981b ; for reviews see Brinkley, 1985; Vorobjev and Nadehzdina, 1987; Karsenti, 1991). By what mechanisms these events are controlled remains largely unknown, but data obtained using phosphoepitope-specific antibodies strongly suggest that phosphorylation of centrosomal proteins plays a major role (Vandré et al., 1984, 1986; Centonze and Borisy, 1990). More direct support for this view stems from the observation that cyclin-dependent kinases (CDKs) enhance the microtubule-nucleation activity of centrosomes at the G2/M transition (Verde et al., 1990, 1992; Buendia et al., 1992) and are involved in promoting centrosome separation (Blangy et al., 1995; Sawin and Mitchison, 1995). Similarly, polo-like kinase 1, a cell cycle regulatory kinase structurally distinct from CDKs, has recently been implicated in centrosome maturation (Lane and Nigg, 1996).The precise role of Nek2 at the centrosome remains to be determined, but it is intriguing that overexpression of this kinase in human cells causes a pronounced splitting of centrosomes. This led us to propose that Nek2-dependent phosphorylation of previously unidentified proteins may cause a loss of centriole–centriole cohesion, and that this event might represent an early step in centrosome separation at the G2/M transition (Fry et al., 1998). With the aim of identifying potential substrates (or regulators) of Nek2, we have now performed a yeast two-hybrid screen, using full-length Nek2 as a bait. We report here the molecular characterization of a novel coiled-coil protein that we call C-Nap1 (for centrosomal Nek2-associated protein 1). C-Nap1 represents a core component of the mammalian centrosome and the first candidate substrate for a member of the NIMA protein kinase family to be identified.     

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.     

Actin-Bundling Protein Isolated from Pollen Tubes of Lily : Biochemical and Immunocytochemical Characterization

A 135-kD actin-bundling protein was purified from pollen tubes of lily (Lilium longiflorum) using its affinity to F-actin. From a crude extract of the pollen tubes, this protein was coprecipitated with exogenously added F-actin and then dissociated from F-actin by treating it with high-ionic-strength solution. The protein was further purified sequentially by chromatography on a hydroxylapatite column, a gel-filtration column, and a diethylaminoethyl-cellulose ion-exchange column. In the present study, this protein is tentatively referred to as P-135-ABP (Plant 135-kD Actin-Bundling Protein). By the elution position from a gel-filtration column, we estimated the native molecular mass of purified P-135-ABP to be 260 kD, indicating that it existed in a dimeric form under physiological conditions. This protein bound to and bundled F-actin prepared from chicken breast muscle in a Ca²⁺-independent manner. The binding of 135-P-ABP to actin was saturated at an approximate stoichiometry of 26 actin monomers to 1 dimer of P-135-ABP. By transmission electron microscopy of thin sections, we observed cross-bridges between F-actins with a longitudinal periodicity of 31 nm. Immunofluorescence microscopy using rhodamine-phalloidin and antibodies against the 135-kD polypeptide showed that P-135-ABP was colocalized with bundles of actin filaments in lily pollen tubes, leading us to conclude that it is the factor responsible for bundling the filaments.Actin filaments, one of the major components of the cytoskeleton, are organized into a highly ordered architecture and are involved in various kinds of cell motility. Their architecture is regulated by several kinds of actin-binding proteins, including cross-linking proteins, severing proteins, end-capping proteins, and monomer-sequestering proteins in animal, protozoan, and yeast cells (Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove and Vancompernolle, 1992). In plant cells the organization of the actin cytoskeleton also changes remarkably during the cell cycle or during developmental processes, and it is suggested that actin-binding proteins are involved in their dynamic change. However, little is known about actin-binding proteins in plant cells.Only a low-M_r actin-binding and -depolymerizing protein, profilin, in white birch (Betula verrucosa; Valenta et al., 1991), maize (Zea mays; Staiger et al., 1993; Ruhlandt et al., 1994), bean (Phaseolus vulgaris; Vidali et al., 1995), tobacco (Nicotiana tabacum; Mittermann et al., 1995), tomato (Lycopersicon esculentum; Darnowski et al., 1996), Arabidopsis (Arabidopsis thaliana; Huang et al., 1996), and lily (Lilium longiflorum; Vidali and Hepler, 1997), and an ADF in lily (Kim et al., 1993), rapeseed (Brassica napus; Kim et al., 1993), and maize (Rozycka et al., 1995; Lopez et al., 1996), have been identified by biochemical or molecular biological means.The native and recombinant forms of these proteins are capable of binding to animal or plant actin (Valenta et al., 1993; Giehl et al., 1994; Ruhlandt et al., 1994; Lopez et al., 1996; Perelroizen et al., 1996; Carlier et al., 1997). Plant profilin expressed in mammalian BHK-21 cells (Rothkegel et al., 1996) or profilin-deficient Dictyostelium discoideum cells (Karakesisoglou et al., 1996) was able to functionally substitute for endogenous profilin in these cells. The introduction of plant profilin into living stamen hair cells by microinjection caused the rapid reduction of the number of actin filaments (Staiger et al., 1994; Karakesisoglou et al., 1996; Ren et al., 1997). These results indicate that plant profilin and ADF share many functional similarities with other eukaryote profilins and ADFs.It is well known that the actin cytoskeleton undergoes dynamic changes in organization during hydration and activation of the vegetative cells of pollen grains (Pierson and Cresti, 1992). Before hydration actin filaments exist as fusiform or spiculate structures (a storage form), but they are rearranged to form a network upon hydration (Heslop-Harrison et al., 1986; Tiwari and Polito, 1988). In the growing pollen tube there are strands or bundles of actin filaments parallel to the long axis (Perdue et al., 1985; Pierson et al., 1986; Miller et al., 1996) that are involved in cytoplasmic streaming (Franke et al., 1972; Mascarenhas and Lafountain, 1972) and transport of vegetative nuclei and generative cells to the growing tip (Heslop-Harrison et al., 1988; Heslop-Harrison and Heslop-Harrison, 1989). Characterization of the function of actin-binding proteins is essential to understanding the regulation of actin organization during the developmental process of pollen. Since only a small number of vacuoles containing proteases develop in pollen grains and pollen tubes at a younger stage, pollen tubes are suitable materials for isolating and biochemically studying actin-binding proteins responsible for organizing actin filaments into various forms.In a previous paper we reported that several components in a crude extract prepared from lily pollen tubes, including a 170-kD myosin heavy chain and 175-, 135-, and 110-kD polypeptides, could be coprecipitated with exogenously added F-actin (Yokota and Shimmen, 1994). We also found that rhodamine-labeled F-actin was tightly bound to the glass surface treated with the fraction containing the 135- and 110-kD polypeptides (Yokota and Shimmen, 1994). These results suggested that either one or both of the 135- and 110-kD polypeptides possesses an F-actin-binding activity. In the present study, we purified the 135-kD polypeptide from lily pollen tubes by biochemical procedures and then characterized its F-actin-binding properties and distribution in the pollen tubes. This protein was able to bundle F-actin isolated from chicken breast muscle and colocalized with actin-filament bundles in pollen tubes. We refer to this protein as P-135-ABP (Plant 135-kD Actin-Bundling Protein).