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.

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

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 Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

2000 ◽  
Vol 11 (1) ◽  
pp. 23-38 ◽  
Author(s):  
Michael J. Lewis ◽  
Benjamin J. Nichols ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman ◽  
Hugh R. B. Pelham
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Early Endosomes ◽  
Internal Structures ◽  
Green Fluorescent

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

scholarly journals Endocytosis Is Crucial for Cell Polarity and Apical Membrane Recycling in the Filamentous Fungus Aspergillus oryzae

2008 ◽  
Vol 8 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Yujiro Higuchi ◽  
Jun-ya Shoji ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Filamentous Fungi ◽  
Aspergillus Oryzae ◽  
Fluorescent Protein ◽  
Calcofluor White ◽  
Transmission Electron Microscopy Analysis ◽  
Physiological Importance ◽  
Protein Receptor ◽  
Green Fluorescent

ABSTRACT Establishing the occurrence of endocytosis in filamentous fungi was elusive in the past mainly due to the lack of reliable indicators of endocytosis. Recently, however, it was shown that the fluorescent dye N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) and the plasma membrane protein AoUapC (Aspergillus oryzae UapC) fused to enhanced green fluorescent protein (EGFP) were internalized from the plasma membrane by endocytosis. Although the occurrence of endocytosis was clearly demonstrated, its physiological importance in filamentous fungi still remains largely unaddressed. We generated a strain in which A. oryzae end4 (Aoend4), the A. oryzae homolog of Saccharomyces cerevisiae END4/SLA2, was expressed from the Aoend4 locus under the control of a regulatable thiA promoter. The growth of this strain was severely impaired, and its hyphal morphology was altered in the Aoend4-repressed condition. Moreover, in the Aoend4-repressed condition, neither FM4-64 nor AoUapC-EGFP was internalized, indicating defective endocytosis. Furthermore, the localization of a secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) was abnormal in the Aoend4-repressed condition. Aberrant accumulation of cell wall components was also observed by calcofluor white staining and transmission electron microscopy analysis, and several genes that encode cell wall-building enzymes were upregulated, indicating that the regulation of cell wall synthesis is abnormal in the Aoend4-repressed condition, whereas Aopil1 disruptants do not display the phenotype exhibited in the Aoend4-repressed condition. Our results strongly suggest that endocytosis is crucial for the hyphal tip growth in filamentous fungi.

scholarly journals Fig1 Facilitates Calcium Influx and Localizes to Membranes Destined To Undergo Fusion during Mating in Candida albicans

2011 ◽  
Vol 10 (3) ◽  
pp. 435-444 ◽  
Author(s):  
Meng Yang ◽  
Alexandra Brand ◽  
Thyagarajan Srikantha ◽  
Karla J. Daniels ◽  
David R. Soll ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Calcium Influx ◽  
Fold Increase ◽  
Gene Encoding ◽  
Content Type ◽  
Daughter Cells ◽  
Green Fluorescent

ABSTRACTFew mating-regulated genes have been characterized inCandida albicans. C. albicans FIG1(CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. InSaccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca2+uptake in mating-competent opaque cells. Uptake was reduced substantially in thefig1Δ null mutant. CaFig1 is therefore involved in Ca2+influx and localizes to membranes that are destined to undergo fusion during mating.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1377
Author(s):  
Adriano M. Gigante ◽  
Francisco Olivença ◽  
Maria João Catalão ◽  
Paula Leandro ◽  
José Moniz-Pereira ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Structural Similarity ◽  
Lytic Cycle ◽  
Specific Lysis ◽  
Double Stranded Dna ◽  
The Family ◽  
N Terminus ◽  
Green Fluorescent

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

2000 ◽  
Vol 11 (11) ◽  
pp. 3873-3883 ◽  
Author(s):  
Maryse Bailly ◽  
Jeffrey Wyckoff ◽  
Boumediene Bouzahzah ◽  
Ross Hammerman ◽  
Vonetta Sylvestre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Epidermal Growth ◽  
Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

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