Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti–endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.Polarity is a fundamental characteristic of most eukaryotic cells, either as a transient phenomenon (e.g., in a moving fibroblast) or a permanent feature (e.g., of an epithelial layer) (Drubin and Nelson, 1996). In epithelial cells, polarity is evident at many levels. At the cell surface, the basolateral and apical membrane domains face different environments (internal and external, respectively) and each membrane contains a distinct set of proteins and lipids (Simons and Fuller, 1985). Acquisition of the fully polarized epithelial phenotype requires assembly of tight and adhering junctions, which serve as barriers separating the apical and basolateral surfaces, and the selective delivery of plasma membrane (PM)¹ molecules and/or their retention at each surface (Rodriguez-Boulan and Powell, 1992; Simons et al., 1992; Wollner and Nelson, 1992).There is great variety among epithelial cells in the way specific PM proteins reach the same or different destinations. For example, kidney-derived MDCK cells sort most apical and basolateral membrane components in the TGN and then export this cargo directly to the “correct” surface (Matter and Mellman, 1994), although a variant line was recently found that delivers Na⁺,K⁺-ATPase to all PM domains randomly and then achieves a predominant basolateral distribution by selective retention (Hammerton et al., 1991; Mays et al., 1995). In other epithelial cells, apical PM proteins are first transported to the basolateral surface and then subsequently transcytosed to the apical domain, with sorting occurring in the endocytic pathway. The extent to which this more circuitous or “indirect” pathway to the apical surface is used depends on the specific protein and cell type (Rodriguez-Boulan and Zurzolo, 1993; Matter and Mellman, 1994). For delivery of apical membrane proteins, hepatocytes in vivo appear to use the indirect pathway exclusively (Bartles et al., 1987; Schell et al., 1992; Maurice et al., 1994), whereas cultured HepG2 cells reportedly deliver selected membrane lipids directly from the TGN to the apical PM (Zaal et al., 1994).The structural information directing membrane proteins through the transcytotic pathway has been elucidated only for the polymeric IgA receptor (pIgA-R). It is a sacrificial receptor whose 103-amino acid cytoplasmic tail contains multiple signals that direct the protein through the secretory pathway and into the transcytotic branch of the endocytic system. pIgA-R''s final destination is the apical membrane where an 80-kD proteolytic fragment of the receptor''s ectodomain is released into the apical milieu. An important difference between the pIgA-R and resident apical PM proteins studied so far is that the latter usually have short cytoplasmic tails with no apparent sorting signal (e.g., aminopeptiase N [APN] and dipeptidyl peptidase IV [DPPIV]), or are glycosyl phosphatidyl inositol (GPI)- anchored (e.g., 5′-nucleotidase [5′NT]). Positive sorting information is present elsewhere in these proteins, e.g., the glycolipid anchor of GPI-proteins (Lisanti and Rodriguez-Boulan, 1990) and the large ectodomains of APN and DPPIV (Vogel et al., 1992, 1995; Weisz et al., 1992), but finer resolution of such global signals has not yet been attained.Many studies have described the membrane compartments involved in the basolateral-to-apical transcytosis of soluble and/or membrane-bound cargo (e.g., Bomsel et al., 1989; Brändli et al., 1990; Hayakawa et al., 1990; van Deurs et al., 1990; van Genderen and van Meer, 1995). Although it is now clear that multiple compartments participate, the existence of stations or carriers that are unique to the transcytotic pathway is still an open question (e.g., Barroso and Sztul, 1994, versus Apodaca et al., 1994), as are the number and location(s) of the sorting site(s) for transcytotic cargo versus cargo destined for the recycling or lysosomal branches of the endocytic system (for reviews see Courtoy, 1993; Sandoval and Bakke, 1994; Gruenberg and Maxfield, 1995; Mostov and Cardone, 1995). The remarkable plasticity of the endocytic system as well as the possibility of real differences in the transport of soluble and membrane cargo may explain some of the apparent paradoxes. Early immuno-EM studies reported that in hepatocytes in vivo, the pIgA-R shares clathrin-coated entry sites with receptors that recycle between the PM and endosomal compartments (asialoglycoprotein receptor [ASGP-R] and mannose-6-phosphate receptor [M6P-R]), but is then segregated from them at the level of peripheral endosomes (called compartment for uncoupling of receptors and ligands) (Geuze et al., 1984). In contrast, the entry site(s) for resident apical proteins transiently present at the basolateral surface is still unknown. However, in liver in situ, newly synthesized DPPIV colocalizes with transcytosing pIgA-R in subapical tubulovesicular structures, suggesting that, at least in these cells, the last steps of transcytosis are common (Barr and Hubbard, 1993). Moreover, transcytotic membranes can be isolated that contain pIgA-R and newly synthesized DPPIV (Barr et al., 1995). Nevertheless, the extent to which different membrane protein classes with a common destination share a common pathway is still unclear.The newly developed WIF-B cell line is an ideal in vitro model for studying PM protein trafficking in polarized hepatocytes (Ihrke et al., 1993; Shanks et al., 1994). WIF-B cells grow in monolayers and acquire a polarized phenotype reminiscent of hepatocytes in vivo; that is, neighboring cells form bile canalicular-like spaces (BC). Each BC is completely sequestered from the surrounding medium as well as the substratum and apical PM proteins are highly concentrated in the BC membrane. Tight junctions prevent mixing of apical and basolateral PM proteins and block diffusion of large molecules such as antibodies from the culture medium into the BC (Ihrke et al., 1993).As a first step toward understanding the transcytotic pathway(s) in WIF-B cells and ultimately in liver, we define here the intracellular trafficking pathways taken by three different classes of membrane proteins that pass through the basolateral membrane: (a) apical PM proteins and pIgA-R; (b) basolaterally recycling receptors; and (c) proteins of the endosomal/lysosomal pathway that cycle through the PM. These proteins were tracked in living WIF-B cells by labeling with specific antibodies at the basolateral surface and determining the distributions of the antigen–antibody complexes at later times. Antibodies to a variety of apical PM proteins and the pIgA-R were specifically and efficiently transcytosed from the basolateral to the apical domain; all passed through a prominent subapical compartment before fusion with the apical PM. In contrast, antibodies to cycling membrane proteins, such as the ASGP-R, transferrin receptor (Tf-R), and M6P-R, and the lysosomal membrane protein lgp120, did not appear to pass through the subapical compartment, but rather were directly transported to the intracellular compartments that contained the highest concentrations of their antigens at steady state. However, antibodies to endolyn-78, another endosomal/lysosomal membrane protein (Croze et al., 1989), appeared transiently in the apical region of the cells before accumulating in lysosomes. Thus, the trafficking of endolyn-78 resembled to some degree the transcytotic route of apical PM proteins and pIgA-R.Our observations verify that transcytosis is a pathway for the delivery of apical PM proteins to the apical surface in WIF-B cells, as is seen in hepatocytes in vivo. Our findings suggest that two successive sorting compartments operate in WIF-B cells. Basolaterally endocytosed proteins pass first through peripheral endosomes, the compartment from which most ASGP-R and transferrin receptor (Tf-R) molecules recycle; from there lysosomal proteins such as lgp120 are directed towards lysosomes whereas transcytotic molecules are sorted out for transport to the apical pole. However, segregation of apical residents from at least one endosomal/lysosomal marker, endolyn-78, appears to occur after these proteins are delivered to an endomembrane compartment in the subapical region.²     

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.     

The Amino-terminal Domain of Desmoplakin Binds to Plakoglobin and Clusters Desmosomal Cadherin–Plakoglobin Complexes

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.Desmosomes are highly organized adhesive intercellular junctions that couple intermediate filaments to the cell surface at sites of cell–cell adhesion (Farquhar and Palade, 1963; Staehelin, 1974; Schwarz et al., 1990; Garrod, 1993; Collins and Garrod, 1994; Cowin and Burke, 1996; Kowalczyk and Green, 1996). Desmosomes are prominent in tissues that experience mechanical stress, such as heart and epidermis, and the disruption of desmosomes or the intermediate filament system in these organs has devastating effects on tissue integrity (Steinert and Bale, 1993; Coulombe and Fuchs, 1994; Fuchs, 1994; McLean and Lane, 1995; Stanley, 1995; Bierkamp et al., 1996; Ruiz et al., 1996). Desmosomes are highly insoluble structures that can withstand harsh denaturing conditions (Skerrow and Matoltsy, 1974; Gorbsky and Steinberg, 1981; Jones et al., 1988; Schwarz et al., 1990). This property of desmosomes facilitated early identification of desmosomal components but has impaired subsequent biochemical analysis of the protein complexes that form between desmosomal components. Ultrastructurally, desmosomes contain a core region that includes the plasma membranes of adjacent cells and a cytoplasmic plaque that anchors intermediate filaments to the plasma membrane. The plaque can be further divided into an outer dense plaque subjacent to the plasma membrane and an inner dense plaque through which intermediate filaments appear to loop.Molecular genetic analysis has revealed that the desmosomal glycoproteins, the desmogleins and desmocollins, are members of the cadherin family of cell–cell adhesion molecules (for review see Buxton et al., 1993, 1994; Cowin and Mechanic, 1994; Kowalczyk et al., 1996). The classical cadherins, such as E-cadherin, mediate calcium-dependent, homophilic cell–cell adhesion (Nagafuchi et al., 1987). The mechanism by which the desmosomal cadherins mediate cell–cell adhesion remains elusive (Amagai et al., 1994; Chidgey et al., 1996; Kowalczyk et al., 1996), although heterophilic interactions have recently been detected between desmogleins and desmocollins (Chitaev and Troyanovsky, 1997). Both classes of the desmosomal cadherins associate with the cytoplasmic plaque protein plakoglobin (Kowalczyk et al., 1994; Mathur et al., 1994; Roh and Stanley, 1995b ; Troyanovsky et al., 1994), which is part of a growing family of proteins that share a repeated motif first identified in the Drosophila protein Armadillo (Peifer and Wieschaus, 1990). This multigene family also includes the desmosomal proteins band 6/plakophilin 1, plakophilin 2a and 2b, and p0071, which are now considered to comprise a subclass of the armadillo family of proteins (Hatzfeld et al., 1994; Heid et al., 1994; Schmidt et al., 1994; Hatzfeld and Nachtsheim, 1996; Mertens et al., 1996).The most abundant desmosomal plaque protein is desmoplakin, which is predicted to be a homodimer containing two globular end domains joined by a central α-helical coiled-coil rod domain (O''Keefe et al., 1989; Green et al., 1990; Virata et al., 1992). Previous studies have demonstrated that the carboxyl-terminal domain of desmoplakin interacts with intermediate filaments (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; Kouklis et al., 1994; Meng et al., 1997), and the amino-terminal domain of desmoplakin is required for desmoplakin localization to the desmosomal plaque (Stappenbeck et al., 1993). Direct evidence supporting a role for desmoplakin in intermediate filament attachment to desmosomes was provided recently when expression of an amino-terminal polypeptide of desmoplakin was found to displace endogenous desmoplakin from cell borders and disrupt intermediate filament attachment to the cell surface in A431 epithelial cell lines (Bornslaeger et al., 1996).The classical cadherins, such as E-cadherin, bind directly to both β-catenin and plakoglobin (Aberle et al., 1994; Jou et al., 1995; for review see Cowin and Burke, 1996). β-Catenin is also an armadillo family member (McCrea et al., 1991; Peifer et al., 1992), and both plakoglobin and β-catenin bind directly to α-catenin (Aberle et al., 1994, 1996; Jou et al., 1995; Sacco et al., 1995; Obama and Ozawa, 1997). α-Catenin is a vinculin homologue (Nagafuchi et al., 1991) and associates with both α-actinin and actin (Knudson et al., 1995; Rimm et al., 1995; Nieset et al., 1997). Through interactions with β- and α-catenin, E-cadherin is coupled indirectly to the actin cytoskeleton, and this linkage is required for the adhesive activity of E-cadherin (Ozawa et al., 1990; Shimoyama et al., 1992). In addition, E-cadherin association with plakoglobin appears to be required for assembly of desmosomes (Lewis et al., 1997), underscoring the importance of E-cadherin in the overall program of intercellular junction assembly. However, the hierarchy of molecular interactions that couple the desmosomal cadherins to the intermediate filament cytoskeleton is largely unknown, although the desmocollin cytoplasmic domain appears to play an important role in recruiting components of the desmosomal plaque (Troyanovsky et al., 1993, 1994). Since desmosomal cadherins form complexes with plakoglobin and because the amino-terminal domain of desmoplakin is required for desmoplakin localization at desmosomes, we hypothesized that the amino-terminal domain of desmoplakin interacts with the desmosomal cadherin– plakoglobin complex.In previous studies, we used L-cell fibroblasts to characterize plakoglobin interactions with the cytoplasmic domains of the desmosomal cadherins and found that the desmosomal cadherins regulate plakoglobin metabolic stability (Kowalczyk et al., 1994) but do not mediate homophilic adhesion (Kowalczyk et al., 1996). To test the ability of the desmoplakin amino-terminal domain to interact with the desmosomal cadherin–plakoglobin complex, we established a series of L-cell lines expressing the desmosomal cadherins in the presence or absence of a desmoplakin amino-terminal polypeptide (DP-NTP).¹ The results indicate that one important function of the desmoplakin amino-terminal domain is to cluster desmosomal cadherin–plakoglobin complexes. In addition, DP-NTP and plakoglobin were found to form complexes that could be co-immunoprecipitated from L-cell lysates. Using the yeast two hybrid system, DP-NTP was found to bind directly to plakoglobin but not Dsg1. These data suggest that plakoglobin couples the amino-terminal domain of desmoplakin to the desmosomal cadherins and that desmoplakin plays an important role in organizing the desmosomal cadherin–plakoglobin complex into discrete plasma membrane domains.     

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.     

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.