scholarly journals The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae

2012 ◽  
Vol 197 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Kamran Anwar ◽  
Robin W. Klemm ◽  
Amanda Condon ◽  
Katharina N. Severin ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Dependent Manner ◽  
In Cells

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER–ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals Inhibiting lysine 353 oxidation of GRP78 by a hypochlorous probe targeting endoplasmic reticulum promotes autophagy in cancer cells

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Junya Ning ◽  
Zhaomin Lin ◽  
Xuan Zhao ◽  
Baoxiang Zhao ◽  
Junying Miao
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Growth ◽  
Cancer Cells ◽  
Cell Fate ◽  
Hypochlorous Acid ◽  
Dependent Manner ◽  
Autophagy Flux ◽  
Post Translational Modifications

Abstract The level of hypochlorous acid (HOCl) in cancer cells is higher than that in non-cancer cells. HOCl is an essential signal for the regulation of cell fate and works mainly through the protein post-translational modifications in cancer cells. However, the mechanism of HOCl regulating autophagy has not been clarified. Here we reported that a HOCl probe named ZBM-H targeted endoplasmic reticulum and induced an intact autophagy flux in lung cancer cells. Furthermore, ZBM-H promoted the binding of GRP78 to AMPK and increased the phosphorylation of AMPK in a dose- and time-dependent manner. GRP78 knockdown inhibited ZBM-H-induced AMPK phosphorylation and ZBM-H-stimulated autophagy. In addition, mass spectrometry combined with point mutation experiments revealed that ZBM-H increased GRP78 activity by inhibiting HOCl-induced lysine 353 oxidation of GRP78. Following ZBM-H treatment in vitro and in vivo, cell growth was significantly inhibited while apoptosis was induced. Nevertheless, exogenous HOCl partially reversed ZBM-H-inhibited cell growth and ZBM-H-induced GRP78 activation. In brief, we found that an endoplasmic reticulum-targeted HOCl probe named ZBM-H, acting through attenuating HOCl-induced GRP78 oxidation, inhibited tumor cell survival by promoting autophagy and apoptosis. Overall, these data demonstrated a novel mechanism of hypochlorous acid regulating autophagy by promoting the oxidation modification of GRP78.

scholarly journals AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Steffen Preissler ◽  
Cláudia Rato ◽  
Ruming Chen ◽  
Robin Antrobus ◽  
Shujing Ding ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Unfolded Protein ◽  
Peptide Dissociation

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

scholarly journals Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (5) ◽  
pp. 2566-2575 ◽  
Author(s):  
T E Blank ◽  
M P Woods ◽  
C M Lebo ◽  
P Xin ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Strong Support ◽  
Constitutive Expression ◽  
Mutant Proteins ◽  
Binding Characteristics ◽  
Isolation And Characterization ◽  
In Cells

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

scholarly journals Functional Conservation of the Glutamine-Rich Domains of Yeast Gal11 and Human SRC-1 in the Transactivation of Glucocorticoid Receptor Tau 1 in Saccharomyces cerevisiae

2007 ◽  
Vol 28 (3) ◽  
pp. 913-925 ◽  
Author(s):  
Dae-Hwan Kim ◽  
Gwang Sik Kim ◽  
Chul Ho Yun ◽  
Young Chul Lee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Mediator Complex ◽  
Dependent Manner ◽  
Activator Proteins ◽  
Functional Conservation

ABSTRACT The yeast Gal11 protein, a component of the Mediator complex, is required for the transcriptional activation of many class II genes as a physiological target of various activator proteins in vivo. In this study, we identified the yeast (Saccharomyces cerevisiae) Mediator complex as a novel coactivator of the transcriptional activity of the glucocorticoid receptor (GR) tau 1 (τ1), the major transcriptional activation domain of the GR. GR τ1 directly interacted with the Mediator complex in vivo and in vitro in a Gal11 module-dependent manner, and the Gal11p subunit interacted directly with GR τ1. Specific amino acid residues within the glutamine-rich (Qr) domain of Gal11p (residues 116 to 277) were essential for its interaction with GR τ1 and GR τ1 transactivity in yeast, as demonstrated by mutational analysis of the Gal11 Qr domain, which is highly conserved among human steroid receptor coactivator (SRC) proteins. A Gal11p variant, mini-Gal11p, comprised of the Mediator association and Qr domains of Gal11p or chimeric mini-Gal11p containing the Qr domain of SRC-1 could potentiate the GR τ1 transactivity in a gal11Δ yeast strain. These results suggest that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.

scholarly journals Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo

2006 ◽  
Vol 397 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Harumi Shimizu ◽  
David Saliba ◽  
Maura Wallace ◽  
Lee Finlan ◽  
Patrick R. R. Langridge-Smith ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Covalent Modification ◽  
High Molecular Mass ◽  
Mutant Form ◽  
Dependent Manner ◽  
Ubiquitination System ◽  
Covalent Adducts ◽  
In Cells

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53F270A) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53R175H or p53F270A also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53R175H or p53F270A unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53F270A:6KR chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53F270A and the p53F270A:6KR chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.

scholarly journals Secretion of Saccharomyces cerevisiae Killer Toxin: Processing of the Glycosylated Precursor

1983 ◽  
Vol 3 (8) ◽  
pp. 1362-1370
Author(s):  
H. Bussey ◽  
D. Saville ◽  
D. Greene ◽  
D. J. Tipper ◽  
K. A. Bostian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Killer Toxin ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Endoproteolytic Cleavage ◽  
Toxin Secretion

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum ( sec18 ), Golgi ( sec7 ), or secretory vesicles ( sec1 ). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl- l -phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post- sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2 . Protoxin stability in kex1 kex2 double mutants indicated the order kex1 → kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1 - and KEX2 -dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

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.

Determinants of triacylglycerol transport from the endoplasmic reticulum to the Golgi in intestine

1997 ◽  
Vol 273 (1) ◽  
pp. G18-G30 ◽  
Author(s):  
N. S. Kumar ◽  
C. M. Mansbach
Keyword(s):  
Endoplasmic Reticulum ◽  
Intestinal Cell ◽  
Liver And Kidney ◽  
Proximal Intestine ◽  
Rat Enterocytes ◽  
Cytosolic Factor ◽  
Intact Rats ◽  
In Cells

The ability of the intestinal cell to export triacylglycerol (TG) is a physiologically regulatable function. The intracellular site where this occurs is unknown, although available evidence suggests that the step between the endoplasmic reticulum (ER) and the Golgi is the most likely. We studied this process in rat enterocytes that were isolated from the proximal intestine. A novel system was developed in which [3H]TG was transported from ER to the Golgi. This process was time, ATP, temperature, and cytosol dependent. The cytosolic factor(s) was heat and trypsin sensitive. TG transport was directly proportional to the amount of added nonradiolabeled acceptor Golgi. The rate of TG transported to the Golgi was the fastest in cells isolated from rats that had been intraduodenally infused in vivo with glyceryltrioleate (TO) plus phosphatidylcholine and slowest in cells isolated from bile-fistulated rats infused with TO in vivo compared with cells from in vivo TO-infused, bile duct intact rats, mimicking the relative transport rates seen in vivo. TG transport in vitro could not be quenched by adding TG emulsions, chylomicrons, liposomes, or guanosine 5'-O-(3-thiotriphosphate). Cytosol from the liver and kidney supported TG transport, but the Golgi from liver or kidney did not accept TG from intestinal ER. We conclude that an intestinally specific, active transport mechanism transports TG from the ER to the Golgi and that this might be a regulatory step in TG export from the intestinal cell.

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