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.     

Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 Å resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.The mechanisms governing the correct timing and localization of cell division is one of the most studied topics in cell biology. In unicellular bacteria like Escherichia coli, cell division occurs at the mid-cell position, away from the chromosomes (1–3). The key step in this process is the appropriate timing and localization of cell division protein FtsZ to the future septum site, followed by polymerization to the Z-ring and sequential recruitment of the divisome components (1, 4). One of the major advances in our understanding of the cell division process and, in particular, of the function of FtsZ came from elucidation of the three-dimensional structure of FtsZ, which showed striking similarity to the eukaryotic protein tubulin, despite very low sequence similarity (5). Such prokaryotic ancestry was later also revealed for the cytoskeletal proteins MreB and Mbl, which belong to the actin family (6, 7), and underscored the notion of generally conserved principles in cytokinesis.Spore-forming Gram-positive Streptomyces bacteria are an important source of clinically useful antibiotics and anticancer agents (8). In these morphologically complex microorganisms, cell division is distinctly different from that in unicellular bacteria in several ways. For one, they are the only known organisms where FtsZ and MreB are both dispensable for growth (9, 10), which makes streptomycetes ideal for the study of cytokinesis. Streptomycetes have a complex life cycle that is mechanistically very similar to filamentous fungi, in producing a mycelium and propagating by sporulation (11, 12). During sporulation, the cell division machinery produces up to 100 septa simultaneously, spaced at around 1 μm, resulting in long chains of uniform and unigenomic spores (10, 13, 14). Besides the simultaneous production of multiple septa, cell division in mycelial actinomycetes also differs from that in other bacteria at the molecular level; actinomycetes lack orthologues of MinC and MinE for septum site localization (15, 16) as well as the nucleoid occlusion system Noc and Z-ring anchoring proteins, such as FtsA and ZipA (1, 6). Instead, several unique protein families have been identified that play a role in the control of cell division, including CrgA and the SsgA-like proteins (17–19). However, molecular details of their mode of action are so far lacking.The SsgA (sporulation of Streptomyces griseus)-like proteins (SALPs)³ are small (around 130–140 residues), actinomycete-specific proteins, which control sporulation-related processes in streptomycetes (17, 20). Streptomyces coelicolor contains seven SALP paralogues (SsgA to SsgG). SsgA and SsgB are essential for sporulation of S. coelicolor (21, 22). SALPs are involved in the control of cell wall-related events, such as septum localization and synthesis, thickening of the spore wall, and autolytic spore separation (17, 20), and SsgA itself directly activates sporulation-specific cell division (22, 23). The morphological complexity of actinomycetes apparently correlates to the number of SALP homologues in each organism, with one paralogue in single spore-forming actinomycetes (e.g. Salinispora or Thermobifida) and up to seven in multispore formers (two in erythromycin producer Saccharopolyspora erythraea, 3–5 in Frankia, and 6–7 in Streptomyces) (17). Most SALPs can be assigned to three subfamilies (SsgA, SsgBG, and SsgDE) based on phylogenetic analysis (17). At present, there are no functional homologues for the SALPs, and structural information is lacking. To advance our understanding of how SALPs function at the molecular level and to provide a structural template for a unique protein family without obvious relatives in any other organism, we selected the single SALP homologue from the thermophilic soil bacterium Thermobifida fusca (a major degrader of plant cell walls used in waste remediation (24)) for detailed structural analysis by x-ray crystallography, as part of our structural genomics program.In this work, we show that SsgB is most likely the archetypical SALP that occurs in morphologically complex actinomycetes, with an evolutionarily conserved function in the control of development. The three-dimensional structure of the SsgB orthologue from T. fusca was determined and revealed significant structural similarity to a eukaryotic family of ssDNA/gRNA-binding proteins. However, the structure and experimental data both suggest that SALPs probably interact with protein ligands through a hydrophobic region on their surface.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Commitment and Effector Phases of the Physiological Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase,and Cyclin-Dependent Kinase Activities

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1β-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.Although interest in the process of physiological cell death has grown enormously in recent years, the mechanism of death has remained enigmatic. While the induction of physiological death in diverse cell types is effected by a wide variety of stimuli, a common morphology, described as apoptosis, ensues in all cases. The commonality of morphology has led to the belief that disparate inducers trigger distinct signaling events which ultimately converge in a common biochemical pathway of death. This hypothesis suggests a division of the biochemical process into upstream events that are specific for individual inducers and downstream steps, comprising the common pathway, which bring about the actual demise of the cell.Since most cell deaths in the nematode Caenorhabditis elegans are induced in a lineage-determined program, the simple pathway of death elucidated in that species (17) is likely to be revealing of downstream steps. Cell death in C. elegans is dependent on the activation of Ced3, a cysteine protease (77, 79), and is inhibited by Ced9 (27). In mammalian cells, a group of Ced3 homologs, termed caspases (1), appears to play a role in virtually all of the physiological cell deaths studied to date. These enzymes cleave on the carboxyl-terminal side of aspartate residues within distinct recognition motifs. Each caspase is synthesized as a proenzyme and activated by cleavage at internal sites, potentially by the same or another caspase class (66, 77). This leads to the notion that caspases function in an ordered cascade, with members of one family activating members of the next. Data consistent with this pattern have been obtained from studies in vitro (41, 60, 65).Of the large family of mammalian caspases, caspase 3 is closely homologous to Ced3 and appears to be involved widely in cell deaths (50, 65). Nonetheless, specific caspases seem not to be associated uniquely with distinct cases of death, and gene-targeting experiments reveal that the absence of a single caspase has extremely limited consequences for cell death responsiveness (38, 39).Similarly, a family of ced9-related death response modulatory genes exists in mammals; the most closely related homolog, bcl-2, is functionally interchangeable with ced9 in the worm (28, 73). These gene products do not function in all mammalian cell deaths (61, 72). Moreover, while the products of some bcl-2 gene family members have death-sparing activity (6, 7), others exert the opposite effect (52, 78).Several cellular proteins, among them poly(ADP-ribose) polymerase (PARP), nuclear lamins, fodrin, and DNA-dependent protein kinase (10, 16, 34), are targets for cleavage by various caspases. In cells spared from death, for example by Bcl-2, these proteolytic events do not occur (9, 13, 18). Still, the cleavage of none of these proteins has been shown to be essential for the cell death response (42, 54, 74). The specific consequences of caspase activation which are lethal are unknown.It may be that the consequence of protease activity is the specific activation of distinct death effectors. We have proposed that essential genes involved in cell division may be critically involved in cell death as well and that the difficulty in identifying distal effector steps genetically reflects the indispensable function of those gene products in cell life (67). Data from several groups have shown that cell cycle catastrophes, the precocious expression of mitosis-like cyclin-dependent histone kinases (Cdks), are associated with a variety of physiological cell deaths and that the inhibition of death by Bcl-2 is associated with alterations in the expression and localization of these Cdk proteins (22, 23, 29, 36, 40, 46, 47, 58, 59, 70).We have taken advantage of the death-sparing activities of Bcl-2 and two viral caspase inhibitors, CrmA and p35 (64, 77), to dissect the mechanism of cell death in two separate cellular paradigms. These studies allow us to draw a generalized skeletal pathway of the death-associated biochemical activities discussed above and demonstrate the requisite involvement of these different classes of activities in a conserved and ordered pathway by which cells die physiologically.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

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.