scholarly journals C-Terminal Signals Regulate Targeting of Glycosylphosphatidylinositol-Anchored Proteins to the Cell Wall or Plasma Membrane in Candida albicans

2008 ◽  
Vol 7 (11) ◽  
pp. 1906-1915 ◽  
Author(s):  
Yuxin Mao ◽  
Zimei Zhang ◽  
Charles Gast ◽  
Brian Wong
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Candida Albicans ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Fluorescent Reporters ◽  
C Terminus ◽  
Peptide Signals ◽  
Gpi Proteins ◽  
Green Fluorescent

ABSTRACT Fungal glycosylphosphatidylinositol (GPI)-anchored proteins localize to the plasma membrane (PM), cell wall (CW), or both. To study signals that regulate PM versus CW targeting in Candida albicans, we (i) fused the N and/or C termini of the GPI CW protein Hwp1p and the GPI PM protein Ecm331p to green fluorescent protein (GFP) and (ii) expressed and localized the resulting fusions. Forty-seven amino acids from the C terminus of Hwp1p were sufficient to target GFP to the CW, and 66 amino acids from the C terminus of Ecm331p were sufficient to target GFP to the PM. Truncation and mutagenesis studies showed that G390 was the ω cleavage site in Ecm331p. Domain exchange and mutagenesis studies showed that (i) the 5 amino acids immediately N-terminal to the ω sites (the ω − 5 to ω − 1 amino acids) played key roles in targeting to the PM or CW; (ii) KK and FE residues at positions ω − 1 and ω − 2, respectively, targeted to the PM and CW; and (iii) a loss of I at position ω − 5 increased PM retention. Small fluorescent reporters can be used to study the peptide signals that regulate PM versus CW targeting of GPI proteins and may be useful for identifying proteins that interact with key targeting signals.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

scholarly journals The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

2005 ◽  
Vol 387 (3) ◽  
pp. 573-584 ◽  
Author(s):  
Sandra MILASTA ◽  
Nicholas A. EVANS ◽  
Laura ORMISTON ◽  
Shelagh WILSON ◽  
Robert J. LEFKOWITZ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Weak Interactions ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Mapk ◽  
Hydroxy Amino ◽  
C Terminus ◽  
Green Fluorescent ◽  
Kinetics Of

The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

scholarly journals A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

scholarly journals Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

2009 ◽  
Vol 20 (22) ◽  
pp. 4856-4870 ◽  
Author(s):  
Eleonora Rolli ◽  
Enrico Ragni ◽  
Julia Calderon ◽  
Silvia Porello ◽  
Umberto Fascio ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Separation ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Cell Envelope ◽  
Phenotypic Analysis ◽  
Neck Size ◽  
Bud Neck ◽  
Green Fluorescent

Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3Δ cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2Δ mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope.

A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane

2001 ◽  
Vol 114 (4) ◽  
pp. 719-726 ◽  
Author(s):  
M.I. Milewski ◽  
J.E. Mickle ◽  
J.K. Forrest ◽  
D.M. Stafford ◽  
B.D. Moyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Binding Motif ◽  
C Terminus ◽  
Polarized Epithelia ◽  
Selective Retention ◽  
Green Fluorescent ◽  
Apical Localization

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370–1394 and 1404–1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

2009 ◽  
Vol 84 (3) ◽  
pp. 1585-1596 ◽  
Author(s):  
Joëlle V. Fritz ◽  
Denis Dujardin ◽  
Julien Godet ◽  
Pascal Didier ◽  
Jan De Mey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Physiology ◽  
Fluorescence Lifetime Imaging ◽  
M Cell ◽  
C Terminus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

2013 ◽  
Vol 12 (6) ◽  
pp. 889-899 ◽  
Author(s):  
Haomiao Ouyang ◽  
Xiaomin Chen ◽  
Yang Lü ◽  
Iain B. H. Wilson ◽  
Guomin Tang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Cellular Localization ◽  
Fluorescent Proteins ◽  
Basic Amino Acid ◽  
Fungal Species ◽  
Content Type ◽  
Gpi Proteins ◽  
Green Fluorescent

ABSTRACTAlthough the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of thecis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined howAspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences fromA. fumigatusMp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half ofAfMp1 was not required for cell wall distribution. Based on our results, thecis-requirements directing localization of GPI proteins inA. fumigatusare different from those in yeast.

scholarly journals Insight into the Antiadhesive Effect of Yeast Wall Protein 1 of Candida albicans

2012 ◽  
Vol 11 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Bruce L. Granger
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Surrounding Medium ◽  
Yeast Cells ◽  
Content Type ◽  
A Cell ◽  
Opportunistic Fungal Pathogen ◽  
Green Fluorescent

ABSTRACTYwp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall ofCandida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to expressYWP1lose adhesion in anin vitroassay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created inC. albicansand harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of theCandidaclade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.

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