Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.     

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.     

Surface localization of the Yps3p protein of Histoplasma capsulatum

The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.     

In vivo processing of nonanchored Yapsin 1 (Yap3p)

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3) was overexpressed in yeast and its processing through the secretory pathway was followed by pulse-labeling and immunoprecipitation studies. In the soluble cell extract, three forms of yapsin 1-87, 74, and 18 kDa-were found. Identification of these forms of yapsin 1 using different antisera suggests that the 87-kDa form is pro-yapsin 1, which is processed into two subunits, alpha (18 kDa) and beta (74 kDa), by cleavage at a loop region not found in traditional aspartic proteases. By use of a temperature-sensitive mutant strain, sec18, the generation of the two subunits was found to occur in the endoplasmic reticulum. An active site-mutated yapsin 1 was not processed into the two subunits, suggesting that this process occurs in an autocatalytic manner.     

Characterization and functional analysis of the β-1,3-glucanosyltransferase 3 of the human pathogenic fungus Paracoccidioides brasiliensis

The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 ( Pb Gel3p) is presented here. Pb Gel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis . The recombinant Pb Gel3p was overexpressed in Escherichia coli , and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of Pb Gel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1 Δ mutant. The results indicated that Pb Gel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.     

An alpha-amylase homologue, aah3, encodes a GPI-anchored membrane protein required for cell wall integrity and morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four alpha-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3(+) resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3(+) deletion, but the ascal wall of aah3Delta cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Delta cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

The presence of an ER exit signal determines the protein sorting upon ER exit in yeast

In yeast, there are at least two vesicle populations upon ER (endoplasmic reticulum) exit, one containing Gap1p (general aminoacid permease) and a glycosylated alpha-factor, gpalphaF (glycosylated proalpha-factor), and the other containing GPI (glycosylphosphatidylinositol)-anchored proteins, Gas1p (glycophospholipid-anchored surface protein) and Yps1p. We attempted to identify sorting determinants for this protein sorting event in the ER. We found that mutant Gas1 proteins that lack a GPI anchor and/or S/T region (serine- and threonine-rich region), two common characteristic features conserved among yeast GPI-anchored proteins, were still sorted away from Gap1p-containing vesicles. Furthermore, a mutant glycosylated alpha-factor, gpalphaGPI, which contains both the GPI anchor and S/T region from Gas1p, still entered Gap1p-containing vesicles, demonstrating that these conserved characteristics do not prevent proteins from entering Gap1p-containing vesicles. gpalphaF showed severely reduced budding efficiency in the absence of its ER exit receptor Erv29p, and this residual budding product no longer entered Gap1p-containing vesicles. These results suggest that the interaction of gpalphaF with Erv29p is essential for sorting into Gap1p-containing vesicles. We compared the detergent solubility of Gas1p and the gpalphaGPI in the ER with that in ER-derived vesicles. Both GPI-anchored proteins similarly partitioned into the DRM (detergent-resistant membrane) in the ER. Based on the fact that they entered different ER-derived vesicles, we conclude that DRM partitioning of GPI-anchored proteins is not the dominant determinant of protein sorting upon ER exit. Interestingly, upon incorporation into the ER-derived vesicles, gpalphaGPI was no longer detergent-insoluble, in contrast with the persistent detergent insolubility of Gas1p in the ER-derived vesicles. We present different explanations for the different behaviours of GPI-anchored proteins in distinct ER-derived vesicle populations.     

An α-Amylase Homologue,aah3, Encodes a GPI-Anchored Membrane Protein Required for Cell Wall Integrity and Morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four α-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3 ⁺ resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3 ⁺ deletion, but the ascal wall of aah3Δ cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Δ cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.     

Synthesis and characterization of the first potent inhibitor of yapsin 1. Implications for the study of yapsin-like enzymes

The potent peptidic inhibitor, Y1, of the basic residue-specific yeast aspartyl protease, yapsin 1, was synthesized and characterized. The inhibitor was based on the peptide sequence of a cholecystokinin(13-33) analog that yapsin 1 cleaved with an efficiency of 5.2 x 10(5) m(-1) s(-1) (Olsen, V., Guruprasad, K., Cawley, N. X., Chen, H. C., Blundell, T. L., and Loh, Y. P. (1998) Biochemistry 37, 2768-2777). The apparent K(i) of Y1 for the inhibition of yapsin 1 was determined to be 64.5 nm, and the mechanism is competitive. Y2 was also developed as an analog of Y1 for coupling to agarose beads. The resulting inhibitor-coupled agarose beads were successfully used to purify yapsin 1 to apparent homogeneity from conditioned medium of a yeast expression system. Utilization of this new reagent greatly facilitates the purification of yapsin 1 and should also enable the identification of new yapsin-like enzymes from mammalian and nonmammalian sources. In this regard, Y1 also efficiently inhibited Sap9p, a secreted aspartyl protease from the human pathogen, Candida albicans, which has specificity for basic residues similar to yapsin 1 and might provide the basis for the prevention or control of its virulence. A single-step purification of Sap9p from conditioned medium was also accomplished with the inhibitor column. N-terminal amino acid sequence analysis yielded two sequences indicating that Sap9p is composed of two subunits, designated here as alpha and beta, similar to yapsin 1.     

A Role for the Karyopherin Kap123p in Microtubule Stability

Several components of the nuclear transport machinery play a role in mitotic spindle assembly in higher eukaryotes. To further investigate the role of this family of proteins in microtubule function, we screened for mutations in Saccharomyces cerevisiae that confer sensitivity to microtubule-destabilizing drugs. One mutant exhibiting this phenotype lacked the gene encoding the karyopherin Kap123p. Analysis of kap123 Δ cells revealed that the drug sensitivity was caused by a defect in microtubule stability and/or assembly. In support of this idea, we demonstrated genetic interactions between the kap123 Δ mutation and mutated alleles of genes encoding α-tubulins and factors controlling microtubule dynamics. Moreover, kap123 Δ cells exhibit defects in spindle structure and dynamics as well as nuclear positioning defects during mitosis. Cultures of kap123 Δ strains are enriched for mononucleated large-budded cells often containing short spindles and nuclei positioned away from the budneck, phenotypes indicative of defects in both cytoplasmic and nuclear microtubules. Finally, we identified a gene, CAJ1 , which when deleted in combination with KAP123 exacerbated the microtubule-related defects of the kap123 Δ mutants. We propose that Kap123p and Caj1p, a member of the Hsp40 family of proteins, together play an essential role in normal microtubule function.     

The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli. MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types. SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis. Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases. CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression. The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway. First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis. Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA. Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall. These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development.     

Posttranslational modifications required for cell surface localization and function of the fungal adhesin Aga1p

Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.     

Modular domain structure in the Candida glabrata adhesin Epa1p,a beta1,6 glucan-cross-linked cell wall protein

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI-anchored glucan-cross-linked cell wall proteins (GPI-CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N-terminal third of the protein contains the ligand-binding domain, and that the GPI anchor is essential both for cross-linking in the cell wall and for Epa1p-mediated adherence. We also found that the C-terminal Ser/Thr-rich domain, characteristic of many GPI-CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C-terminal Ser/Thr-rich region was absolutely required to project the N-terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr-rich domain of Epa1p and, presumably, of other related GPI-CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.     

AgrD-dependent quorum sensing affects biofilm formation, invasion, virulence and global gene expression profiles in Listeria monocytogenes

The Listeria monocytogenes Agr peptide-sensing system has been analysed by creating a deletion mutant in agrD , the structural gene for the putative quorum-sensing peptide. The Δ agrD mutant displayed significantly reduced biofilm formation, a defect which could be restored by genetic or physical complementation. A reduced invasion of Caco-2 intestinal epithelial cells was observed for the Δ agrD mutant while phagocytosis by THP-1 macrophages was unaffected. Additionally, the level of internalin A (InlA) in the cell wall was decreased in the Δ agrD mutant. Expression profiling of virulence genes ( hlyA , actA , plcA , prfA and inlA ) identified a finely tuned regulation which resulted in an impaired virulence response in the Δ agrD mutant. The mutant is also significantly attenuated for virulence in mice, as revealed by bioluminescent in vivo imaging. On day 3 post infection, systemic dissemination to livers and spleens had occurred for the wild type, whereas the Δ agrD mutant remained localized to the liver. Microarray analysis identified 126 and 670 genes as significantly regulated in exponential and stationary phase respectively. The results presented here suggest that peptide sensing plays an important role in the biology of L. monocytogenes , with relevant phenotypes in both the saprophytic and parasitic lifecycles.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.