scholarly journals mBet3p is required for homotypic COPII vesicle tethering in mammalian cells

2006 ◽  
Vol 174 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Sidney Yu ◽  
Ayano Satoh ◽  
Marc Pypaert ◽  
Karl Mullen ◽  
Jesse C. Hay ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Transitional Endoplasmic Reticulum ◽  
Large Complex ◽  
Copii Vesicle ◽  
Copii Vesicles

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

scholarly journals Remodeling of secretory compartments creates CUPS during nutrient starvation

2014 ◽  
Vol 207 (6) ◽  
pp. 695-703 ◽  
Author(s):  
David Cruz-Garcia ◽  
Amy J. Curwin ◽  
Jean-François Popoff ◽  
Caroline Bruns ◽  
Juan M. Duran ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Nitrogen Starvation ◽  
Growth Conditions ◽  
Vesicle Tethering ◽  
Copii Vesicle ◽  
Peripheral Membrane Protein ◽  
Er Exit Sites ◽  
Endosomal Membranes

Upon starvation, Grh1, a peripheral membrane protein located at endoplasmic reticulum (ER) exit sites and early Golgi in Saccharomyces cerevisiae under growth conditions, relocates to a compartment called compartment for unconventional protein secretion (CUPS). Here we report that CUPS lack Golgi enzymes, but contain the coat protein complex II (COPII) vesicle tethering protein Uso1 and the Golgi t-SNARE Sed5. Interestingly, CUPS biogenesis is independent of COPII- and COPI-mediated membrane transport. Pik1- and Sec7-mediated membrane export from the late Golgi is required for complete assembly of CUPS, and Vps34 is needed for their maintenance. CUPS formation is triggered by glucose, but not nitrogen starvation. Moreover, upon return to growth conditions, CUPS are absorbed into the ER, and not the vacuole. Altogether our findings indicate that CUPS are not specialized autophagosomes as suggested previously. We suggest that starvation triggers relocation of secretory and endosomal membranes, but not their enzymes, to generate CUPS to sort and secrete proteins that do not enter, or are not processed by enzymes of the ER–Golgi pathway of secretion.

scholarly journals Inositol acylation of glycosylphosphatidylinositols in the pathogenic fungus Cryptococcus neoformansz and the model yeast Saccharomyces cerevisiae

1999 ◽  
Vol 340 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Sarah P. FRANZOT ◽  
Tamara L. DOERING
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Mammalian Cells ◽  
Pathogenic Fungus ◽  
Sugar Residue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
Life Threatening

Cryptococcus neoformans, an opportunistic fungus responsible for life-threatening infection in immunocompromised patients, is able to synthesize glycosylphosphatidylinositol (GPI) structures. Radiolabelling experiments in vitro with the use of a cryptococcal cell-free system showed that the pathway begins as in other eukaryotes, with the addition of N-acetylglucosamine to phosphatidylinositol, followed by deacetylation of the sugar residue. The third step, acylation of the inositol ring, seemed to involve a fatty acid other than palmitate, in contrast with previous findings in Saccharomyces cerevisiae and mammalian GPI pathways. A systematic study of inositol acylation in C. neoformans and S. cerevisiae showed that both organisms used a variety of fatty acids in this step; these were transferred directly from acyl-CoA to inositol without modification. However, the specificity of fatty acid utilization was quite distinct in the two fungi, with the pathogen being substantially more restrictive. In mammalian cells fatty acids added exogenously as acyl-CoAs are not transferred directly to inositol. These results suggest significant differences in the GPI biosynthetic pathway between mammalian and C. neoformans cells that could represent targets for anti-cryptococcal therapy.

scholarly journals Characterization of sphingosine kinase (SK) activity in Saccharomyces cerevisiae and isolation of SK-deficient mutants

1998 ◽  
Vol 332 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Margaret M. LANTERMAN ◽  
Julie D. SABA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Growth Regulation ◽  
Sphingosine Kinase ◽  
Heat Sensitivity ◽  
Sphingoid Bases ◽  
Yeast Saccharomyces Cerevisiae ◽  
Abnormal Growth

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 °C. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 °C, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

scholarly journals Hypothesis: cell volume limits cell divisions.

2006 ◽  
Vol 53 (4) ◽  
pp. 833-835 ◽  
Author(s):  
Tomasz Biliński ◽  
Grzegorz Bartosz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Volume ◽  
Mammalian Cells ◽  
Somatic Cells ◽  
Large Cell ◽  
Yeast Cells ◽  
Replicative Aging ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Divisions

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.

scholarly journals Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels

2016 ◽  
Vol 27 (10) ◽  
pp. 1635-1649 ◽  
Author(s):  
Indrani Mukherjee ◽  
Charles Barlowe
Keyword(s):  
Secretory Pathway ◽  
Cytosolic Calcium ◽  
Live Cells ◽  
Rab Gtpase ◽  
Multicopy Suppressor ◽  
Vesicle Tethering ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Polytopic Membrane Protein

SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca2+/Mn2+transporter and suggests that Sly41 influences cellular Ca2+and Mn2+homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca2+concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca2+on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport.

scholarly journals Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

2004 ◽  
Vol 167 (6) ◽  
pp. 997-1003 ◽  
Author(s):  
Dalu Xu ◽  
Jesse C. Hay
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Secretory Pathway ◽  
Golgi Stack ◽  
Golgi Membranes ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Copii Vesicles

What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

scholarly journals Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.

1987 ◽  
Vol 105 (6) ◽  
pp. 2905-2914 ◽  
Author(s):  
P Bird ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Influenza Virus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Signal Sequence ◽  
Carboxypeptidase Y ◽  
Influenza Virus Hemagglutinin

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2298-2305
Author(s):  
G L Temeles ◽  
D DeFeo-Jones ◽  
K Tatchell ◽  
M S Ellinger ◽  
E M Scolnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Cross Reactivity ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Ras Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
In The Wild

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

scholarly journals Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2298-2305 ◽  
Author(s):  
G L Temeles ◽  
D DeFeo-Jones ◽  
K Tatchell ◽  
M S Ellinger ◽  
E M Scolnick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Cross Reactivity ◽  
Binding Activity ◽  
In Vitro Translation ◽  
Ras Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Genes ◽  
In The Wild

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

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