Export of major cell surface proteins is blocked in yeast secretory mutants

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.

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Roles ofCandida albicansDfg5p and Dcw1p Cell Surface Proteins in Growth and Hypha Formation

Eukaryotic Cell ◽

10.1128/ec.2.4.746-755.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 746-755 ◽

Cited By ~ 86

Author(s):

Elisabetta Spreghini ◽

Dana A. Davis ◽

Ryan Subaran ◽

Michelle Kim ◽

Aaron P. Mitchell

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Insertional Mutagenesis ◽

Surface Proteins ◽

Alkaline Media ◽

Specific Gene ◽

External Signal ◽

Cell Surface Proteins ◽

Hypha Formation ◽

Conditional Expression

TheCandida albicanscell wall participates in both growth and morphological transitions between yeast and hyphae. Our studies here focus on Dfg5p and Dcw1p, two similar proteins with features of glycosylphosphatidylinositol-linked cell surface proteins. Mutants lacking Dfg5p are defective in alkaline pH-induced hypha formation; mutants lacking Dcw1p have no detected hypha formation defect. Both homozygote-triplication tests and conditional expression strategies indicate thatdfg5anddcw1mutations are synthetically lethal. Therefore, Dfg5p and Dcw1p share a function required for growth. Epitope-tagged Dfg5p, created through an insertional mutagenesis strategy, is found in cell membrane and cell wall extract fractions, and endoglycosidase H digestion shows that Dfg5p undergoes N-linked mannosylation. Surprisingly, Dfg5p is required for expression of the hypha-specific geneHWP1in alkaline media. Because Dfg5p is a cell surface protein, it is poised to generate or transmit an external signal required for the program of hypha-specific gene expression.

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Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris

Genetics ◽

10.1093/genetics/iyab029 ◽

2021 ◽

Cited By ~ 3

Author(s):

José F Muñoz ◽

Rory M Welsh ◽

Terrance Shea ◽

Dhwani Batra ◽

Lalitha Gade ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Genome Instability ◽

Chromosomal Rearrangements ◽

Multidrug Resistant ◽

Antifungal Drugs ◽

Surface Proteins ◽

Cell Wall Proteins ◽

Candida Auris ◽

Cell Surface Proteins

Abstract Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

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Interaction of Lp(a) with Plasminogen Binding Sites on Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653797 ◽

1995 ◽

Vol 73 (03) ◽

pp. 458-465 ◽

Cited By ~ 42

Author(s):

Lindsey A Miles ◽

Gunther M Fless ◽

Angelo M Scanu ◽

Patricia Baynham ◽

Matthew T Sebald ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Binding Sites ◽

Surface Proteins ◽

Time Dependent ◽

Temperature Sensitive ◽

U937 Cells ◽

Cell Surface Proteins ◽

Sensitive Characteristics ◽

Lipoprotein Binding

SummaryLp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 μM), but Lp(a) bound to fewer sites (̴10-fold less). Both gangliosides and cell surface proteins with car- boxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).

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Role of SrtA in Pathogenicity of Staphylococcus lugdunensis

Microorganisms ◽

10.3390/microorganisms8121975 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1975

Author(s):

Muzaffar Hussain ◽

Christian Kohler ◽

Karsten Becker

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Biofilm Formation ◽

Surface Proteins ◽

Transcription Level ◽

Coagulase Negative Staphylococci ◽

Staphylococcus Lugdunensis ◽

Cell Surface Proteins ◽

Mutant Strains

Among coagulase-negative staphylococci (CoNS), Staphylococcus lugdunensis has a special position as causative agent of aggressive courses of infectious endocarditis (IE) more reminiscent of IEs caused by Staphylococcus aureus than those by CoNS. To initiate colonization and invasion, bacterial cell surface proteins are required; however, only little is known about adhesion of S. lugdunensis to biotic surfaces. Cell surface proteins containing the LPXTG anchor motif are covalently attached to the cell wall by sortases. Here, we report the functionality of Staphylococcus lugdunensis sortase A (SrtA) to link LPXTG substrates to the cell wall. To determine the role of SrtA dependent surface proteins in biofilm formation and binding eukaryotic cells, we generated SrtA-deficient mutants (ΔsrtA). These mutants formed a smaller amount of biofilm and bound less to immobilized fibronectin, fibrinogen, and vitronectin. Furthermore, SrtA absence affected the gene expression of two different adhesins on transcription level. Surprisingly, we found no decreased adherence and invasion in human cell lines, probably caused by the upregulation of further adhesins in ΔsrtA mutant strains. In conclusion, the functionality of S. lugdunensis SrtA in anchoring LPXTG substrates to the cell wall let us define it as the pathogen’s housekeeping sortase.

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Control of Amino Acid Permease Sorting in the Late Secretory Pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST78

Genetics ◽

10.1093/genetics/147.4.1569 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1569-1584 ◽

Cited By ~ 14

Author(s):

Kevin J Roberg ◽

Stephen Bickel ◽

Neil Rowley ◽

Chris A Kaiser

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Secretory Pathway ◽

Protein Transport ◽

Secretory Vesicle ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Amino Acid Permease ◽

New Genes ◽

Amino Acid Permeases

Abstract The SEC13 gene was originally identified by temperature-sensitive mutations that block all protein transport from the ER to the Golgi. We have found that at a permissive temperature for growth, the sec13-1 mutation selectively blocks transport of the nitrogen-regulated amino acid permease, Gaplp, from the Golgi to the plasma membrane, but does not affect the activity of constitutive permeases such as Hip1p, Can1p, or Lyp1p. Different alleles of SEC13 exhibit different relative effects on protein transport from the ER to the Golgi, or on Gap1p activity, indicating distinct requirements for SEC13 function at two different steps in the secretory pathway. Three new genes, LST4, LST7, and LSTB, were identified that are also required for amino acid permease transport from the Golgi to the cell surface. Mutations in LST4 and LST7 reduce the activity of the nitrogen-regulated permeases Gap1p and Put4p, whereas mutations in LST8 impair the activities of a broader set of amino acid permeases. The LST8 gene encodes a protein composed of WD-repeats and has a close human homologue. The LST7 gene encodes a novel protein. Together, these data indicate that SEC13, LST4, LST7, and LST8 function in the regulated delivery of Gap1p to the cell surface, perhaps as components of a post-Golgi secretory-vesicle coat.

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What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140300 ◽

1995 ◽

Vol 53 ◽

pp. 786-787

Author(s):

Watt W. Webb

Keyword(s):

Cell Surface ◽

Lipid Membranes ◽

Surface Proteins ◽

Protein Diffusion ◽

Coated Pits ◽

Cell Surface Proteins ◽

Membrane Heterogeneity ◽

Fluorescence Photobleaching Recovery ◽

Photobleaching Recovery ◽

Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

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