Sequential Loading of Saccharomyces cerevisiae Ku and Cdc13p to Telomeres

Ku is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C_1–3(A/T)G_1–3 telomeric DNA repeats plus single-stranded TG_1–3 telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres.DNA damages in the form of double-stranded breaks (DSBs)⁴ compromise the integrity of genomes. Failure in repairing or mis-repairing double-stranded breaks can lead to chromosome instability and eventually cell death or cancer (1). Double-stranded breaks are repaired by two main pathways, the homologous recombination and nonhomologous DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to bind to the DNA ends to initiate the repair pathway (2). Upon binding, Ku then recruits a series of repair enzymes to join the broken ends (2). Ku is a heterodimeric protein composed of 70- and ∼80-kDa subunits. In Saccharomyces cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical configuration of the broken ends could be very diverse on DSBs, Ku binds to double-stranded ends in a sequence- and energy-independent manner. It is capable of binding to DNA ends with blunt 3′-overhangs or 5′-overhangs as well as double-stranded DNA with nicks, gaps, or internal loops (3–7). However, Ku does not have high affinity to single-stranded DNA. The crystal structure of human Ku heterodimer indicates that it forms a ring structure that encircles duplex DNA (7). This unique structure feature enables Ku to recognize DNA ends and achieves its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to be a component of telomeric protein-DNA complex in yeast and mammals (8–10). Telomeres are terminal structures of chromosomes composed of short tandem repeated sequences (11, 12). Mutation of YKU70 or YKU80 causes defects in telomere structure (13–15), telomere silencing (16–19), and replication timing of telomeres (20). The function of yeast Ku (Yku) on telomeres could mediate through protein-protein interaction with Sir4p or protein-RNA interaction with Tlc1 RNA (21, 22). For example, through the interaction with Sir4p, Yku selectively affects telomeres silencing but not the silent mating type loci (17). Yku could also bind to telomerase Tlc1 RNA for telomere length maintenance (22). Judged by the DNA binding activity of Yku, it is reasonable to suggest that it may bind directly to telomeric DNA. Indeed, it was shown that human Ku is capable of binding directly to telomeric DNA in vitro (15). Moreover, because the deletion of SIR4 in budding yeast (23) or Taz1 in fission yeast (24) does not abolish the association of Ku with chromosomal ends, this suggests that Ku might bind directly to telomeric DNA in cells. However, because yeast telomeres have a short 12–14-mer single-stranded tail (25), it is uncertain whether Yku could pass the single-stranded region to reach its binding site. The direct binding of Yku to telomeric DNA has not been experimentally determined.In contrast to double-stranded breaks, the ends of linear chromosomes are not recognized by repair enzymes as DNA damage. In S. cerevisiae, Cdc13p is the single-stranded TG_1–3 DNA-binding protein that enables cells to differentiate whether the ends of a linear DNA are telomeres or broken ends (26–29). Thus, although the mechanism of how cells prevent the activation of DSB repair pathway in telomere is unclear, it is likely that binding of Cdc13p to telomeres might inhibit the initiation of DNA damage response by the Ku protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA sequences to mimic telomere structure, we showed that Yku binds directly to this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our results also showed that Yku loaded to a de novo synthesized telomere earlier than Cdc13p in vivo. These results support the direct binding of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block the activation of DSB repair pathway on telomeres.     

Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.     

Characterization of the DNA binding features of Saccharomyces castellii Cdc13p

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand. Moreover, the minimal binding site for scasCdc13p is the octamer 5'-GTGTCTGG-3' of the S.castellii telomeric sequence. The scasCdc13p displayed a high affinity binding, where four individual nucleotide residues were found to be of most importance for the sequence specificity. Nonetheless, scasCdc13p binds the telomeric repeats from various other species, including the human. In spite of considerable divergence in telomere repeat length and sequence between these species, a conserved Cdc13p binding motif was detected. Among the budding yeasts this conserved Cdc13p binding site overlaps the Rap1p binding site. Together, these data implicate scasCdc13p as a telomere end-binding protein with a potential role in the regulation of telomere maintenance in vivo. Moreover, the results suggest that Rap1p and Cdc13p act together to preserve the conserved core present within the otherwise highly divergent btelomeric sequences among a wide variety of yeasts.     

Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae

Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

Binding and partial denaturing of G-quartet DNA by Cdc13p of Saccharomyces cerevisiae

The protein Cdc13p binds telomeres in vivo and is essential for the maintenance of the telomeres of Saccharomyces cerevisiae. In addition, Cdc13p is known to bind single-stranded TG(1-3) DNA in vitro. Here we have shown that Cdc13p also binds DNA quadruplex, G-quartet, formed by TG(1-3) DNA. Moreover, the binding of Cdc13p causes a partial denaturing of the G-quartet DNA. Formation of DNA quadruplexes may involve the intermolecular association of TG(1-3) DNA and inhibit the extension of telomeres by telomerase. Thus, our finding suggests that Cdc13p may disrupt telomere association and facilitate telomere replication.     

Elaboration, Diversification and Regulation of the Sir1 Family of Silencing Proteins in Saccharomyces

This study quantifies the effects of naturally occurring X-linked variation on immune response in Drosophila melanogaster to assess associations between immunity genotypes and innate immune response. We constructed a set of 168 X-chromosomal extraction lines, incorporating X chromosomes from a natural population into co-isogenic autosomal backgrounds, and genotyped the lines at 88 SNPs in 20 X-linked immune genes. We find that genetic variation in many of the genes is associated with immune response phenotypes, including bacterial load and immune gene expression. Many of the associations act in a sex-specific or sexually antagonistic manner, supporting the theory that with the selective pressures facing genes on the X chromosome, sexually antagonistic variation may be more easily maintained.THE deep evolutionary conservation of many specific genes in innate immunity underscores the potent forces of natural selection maintaining this vital function. While it is widely accepted as the ancestral form of immune response, its role in the activation of adaptive immune response further motivates investigation into variation in its function (Medzhitov and Janeway 1997). Drosophila has been used as a valuable model organism to identify and characterize functions of the components of innate immune pathways as well as the evolutionary patterns present among the genes comprising these pathways (reviewed in Brennan and Anderson 2004; Irving et al. 2004; Ferrandon et al. 2007). The humoral response, resulting in the production of antimicrobial peptides in response to bacterial or fungal infection, relies mainly on Toll and imd signal transduction pathways, both of which are highly homologous to pathways in mammalian immunity (reviewed in Kimbrell and Beutler 2001). The cellular component, on the other hand, incorporates phagocytic engulfment as well as melanization and encapsulation of infecting particles. While less well defined in the Drosophila model, portions of other systems also appear to affect the effectiveness of immune response, including JAK/STAT and JNK signaling pathways, hematopoesis, and iron metabolism.Population genetic analysis can be used to determine whether sequence polymorphism and divergence patterns among Drosophila genes in innate immune pathways are consistent with signatures of selection acting within and between species of flies. If, for example, the innate immune pathways are involved in an evolutionary “arms race” with pathogenic organisms, genes in these pathways would be expected to show signs of positive selection driven by evolutionary pressure to counter virulence mechanisms of invading microbes. When signs of selection (as inferred from sequence comparisons within Drosophila simulans populations and between D. simulans and D. melanogaster) in immune genes and nonimmune genes were evaluated, immune genes as a group were found to have higher K_A/K_S ratios than nonimmune genes, providing evidence for elevated adaptive evolution (Schlenke and Begun 2003). Since receptor, effector, and signaling proteins function in different portions of the immune response pathways, these may be exposed to differing levels of contact with invading microbes and may display nonuniform levels of functional redundancy or pleiotropy. Thus, genes from different functional groups may be exposed to distinct selective pressures. Antimicrobial peptides, which might be expected to encounter unique selective pressures due to their direct interactions with invading microbes, have shown little sign of positive selection, bearing low levels of amino acid divergence (Clark and Wang 1997; Date et al. 1998; Ramos-Onsins and Aguadé 1998; Lazzaro and Clark 2003). Furthermore, sequence analyses of immune-related receptors have shown evidence for purifying selection in peptidoglycan recognition proteins (PGRPs), while others, including some scavenger receptors (SRs), appear to be rapidly evolving under pressures consistent with positive selection (Jiggins and Hurst 2003; Lazzaro 2005). On a deeper evolutionary timescale, sequence comparisons between immune genes in multiple Drosophila species (based on full-genome sequence data) have shown striking differences among functional groups of immune genes, with recognition molecules showing much more positive selection than either signaling or effector genes (Sackton et al. 2007).Beyond using sequence data and the analysis of polymorphism and divergence to infer levels and modes of selection that have previously acted on immune genes (either individually or in functional groups), other studies have investigated correlations between autosomal variation in genotype and immune response phenotype in natural populations of Drosophila (Lazzaro et al. 2004, 2006). These experiments tested associations between naturally occurring genetic variation in immune-related genes and postinfection bacterial load. In these studies, genetic variation in many of the immune genes was found to associate significantly with one or more of the bacterial load phenotypes. Specifically, polymorphisms in autosomal genes encoding recognition and signaling proteins (but not antimicrobial peptides) associate consistently with bacterial load phenotypes, suggesting that not all functional classes of immune-related genes harbor equally influential genetic variation.The focus of this study is X-linked immune genes, which may be under unique regulatory and selective pressures simply because they are hemizygous in males, are dosage compensated, and face elevated influence of random genetic drift due to their smaller effective population size. As a consequence, the X chromosome should favor the more rapid fixation of beneficial recessive alleles and more rapid loss of harmful recessive alleles compared to the autosomes (Charlesworth et al. 1987; Singh et al. 2008). Thus, with different selective pressures compared to autosomal genes, X-linked immunity genes are expected to bear different standing levels of variation, and segregating polymorphisms in these genes may have different impacts on phenotype.Different exposures of X-linked genes to selection in males and females can also contribute to sexual dimorphism. Rice (1984) suggested that X-linked sexually antagonistic alleles may more freely influence sexually dimorphic traits than can those on autosomes. In fact, the X chromosome appears to favor the maintenance of sexually antagonistic variation (Gibson et al. 2002); if a given allele is slightly deleterious in one sex, it may be maintained in the population by being beneficial to the other sex. Immune-related genes may be particularly prone to bearing sexual dimorphism in Drosophila, since males and females have been shown to have different evolutionary optima for energetic expenditure on immune response, and thus their respective immune responses may differ on the basis of conditions such as food or reproductive resource availability (McKean and Nunney 2001, 2005). If sexually antagonistic traits are responsible for some of the observed sexual dimorphism, variation in X-linked genes could contribute to phenotypic differences, and so X-linked variation in immune genes could face unique selective pressures.In this report we investigate the standing levels of variation in X-linked immune genes in natural populations of D. melanogaster and quantify the impacts of that variation on immune response phenotypes. We genotyped 168 lines at single-nucleotide polymorphisms (SNPs) across 20 X-linked immunity loci and quantified postinfection bacterial load and immune gene expression phenotypes. We found significant variation across the lines for bacterial load after infection, and we were able to identify polymorphisms in immune-related genes that associate with immune response phenotypes individually and in interacting pairs of SNPs. Additionally, some of the genetic variation was found to associate with a sex difference in immune competence, with alleles acting in either a sex-specific or a sexually antagonistic manner. This provides evidence for X-linked genetic variation in immune-related loci associating with both phenotypic variation among lines and sex differences in these phenotypes.     

At Short Telomeres Tel1 Directs Early Replication and Phosphorylates Rif1

The replication time of Saccharomyces cerevisiae telomeres responds to TG_1–3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

The role of Stn1p in Saccharomyces cerevisiae telomere capping can be separated from its interaction with Cdc13p

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.     

Renaturation and stabilization of the telomere-binding activity of Saccharomyces Cdc13(451-693)p by L-arginine.

Production of recombinant proteins can be valuable in studying their biological functions. However, recombinant proteins expressed in Escherichia coli sometimes form undesirable insoluble aggregates. Solubilization and renaturation of these aggregates becomes a problem that one needs to solve. Here we used recombinant Cdc13(451-693)p as example to show the presence of l-arginine during renaturation greatly enhanced the renaturation efficiency. Cdc13p is the single-stranded telomere-binding protein of yeast Saccharomyces cerevisiae. The telomere-binding domain has been mapped within amino acids 451-693 of Cdc13p, Cdc13(451-693)p. Recombinant Cdc13(451-693)p was expressed in E. coli as insoluble protein aggregates. Purification of insoluble Cdc13(451-693)p was achieved by denaturing the protein with 6 M guanidine-HCl and followed by Ni-nitrilotriacetic acid agarose column chromatography. Renaturation of Cdc13(451-693)p to the active form was achieved by dialyzing denatured protein in the presence of l-arginine. Moreover, the presence of l-arginine was also helped in maintaining the telomere-binding activity of Cdc13(451-693)p. Taking together, l-arginine might have a general application in renaturation of insoluble aggregates.     

In Vivo Assay for the Synthesis of Hydroxyproline-rich Proteins

Simple methods are described for following in vivo the rate of peptidylproline hydroxylation and for determining what proportion of the total proline incorporated into protein is hydroxylated.     

Evoked Release of Proteins from Central Neurons In Vivo

Push-pull cannulae were implanted in both substantiae nigrae and caudate nuclei of the halothane-anesthetized cat. The release of total protein, acetylcho-linesterase, and nonspecific cholinesterases was examined. Following direct application of potassium to one substantia nigra, changes occurred in the local release of total protein and acetylcholinesterase, but not nonspecific cholinesterases; changes also were observed in both caudate nuclei and the contralatera/ substantia nigra. The local evoked release of acetylcholinesterase and of total protein differed in the extent to which they were calcium-dependent. Control studies suggest that release of these compounds, both spontaneous and evoked, is related, at least in part, to neuronal activity. The significance of the neuronal release of proteins is discussed.     

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.     

Probing the Molecular Environment of Membrane Proteins In Vivo

The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo. Cub, the C-terminal half of Ub, was attached to Sec63p, and Nub, the N-terminal half of Ub, was attached to a selection of differently localized proteins of the yeast Saccharomyces cerevisiae. The efficiency of the Nub and Cub reassembly to the quasi-native Ub reflects the proximity between Sec63-Cub and the Nub-labeled proteins. By using a modified Ura3p as the reporter that is released from Cub, the local concentration between Sec63-Cub-RUra3p and the different Nub-constructs could be translated into the growth rate of yeast cells on media lacking uracil. We show that Sec63p interacts with Sec62p and Sec61p in vivo. Ssh1p is more distant to Sec63p than its close sequence homologue Sec61p. Employing Nub- and Cub-labeled versions of Ste14p, an enzyme of the protein isoprenylation pathway, we conclude that Ste14p is a membrane protein of the ER. Using Sec63p as a reference, a gradient of local concentrations of different t- and v-SNARES could be visualized in the living cell. The RUra3p reporter should further allow the selection of new binding partners of Sec63p and the selection of molecules or cellular conditions that interfere with the binding between Sec63p and one of its known partners.     

Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.