scholarly journals Continuous Equilibration of Phosphatidylcholine and Its Precursors between Endoplasmic Reticulum and Mitochondria in Yeast

2003 ◽  
Vol 14 (5) ◽  
pp. 2142-2150 ◽  
Author(s):  
Anton I.P.M. de Kroon ◽  
Martijn C. Koorengevel ◽  
Tom A.M Vromans ◽  
Ben de Kruijff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Subcellular Fractionation ◽  
Unknown Mechanism ◽  
Cytosolic Factors

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

2003 ◽  
Vol 77 (2) ◽  
pp. 1578-1583 ◽  
Author(s):  
Zhenming Xu ◽  
Jinah Choi ◽  
Wen Lu ◽  
Jing-hsiung Ou
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Half Life ◽  
Proteasome Inhibitors ◽  
Subcellular Fractionation ◽  
Biological Properties ◽  
F Protein ◽  
Proteasome Pathway

ABSTRACT Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

Recycling of the yeast v-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec20p

1998 ◽  
Vol 111 (11) ◽  
pp. 1507-1520 ◽  
Author(s):  
W. Ballensiefen ◽  
D. Ossipov ◽  
H.D. Schmitt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Subcellular Fractionation ◽  
Hybrid Protein ◽  
Wild Type ◽  
Alpha Factor ◽  
Membrane Compartments

Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an alpha-factor-tagged Sec22 protein (Sec22-alpha) was employed. Only on reaching the late Golgi, can alpha-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-alpha is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-alpha is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-alpha is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-alpha rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-alpha and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.

scholarly journals Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (11) ◽  
pp. 5021-5037 ◽  
Author(s):  
Mark Trautwein ◽  
Jörn Dengjel ◽  
Markus Schirle ◽  
Anne Spang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Short Range ◽  
Small Gtpase ◽  
Polya Tail ◽  
Cellular Processes ◽  
Golgi Membranes ◽  
The Stability ◽  
Ash1 Mrna

The small GTPase Arf1p is involved in different cellular processes that require its accumulation at specific cellular locations. The recruitment of Arf1p to distinct points of action might be achieved by association of Arf1p with different proteins. To identify new interactors of Arf1p, we performed an affinity chromatography with GTP- or GDP-bound Arf1p proteins. A new interactor of Arf1p-GTP was identified as Pab1p, which binds to the polyA-tail of mRNAs. Pab1p was found to associate with purified COPI-coated vesicles generated from Golgi membranes in vitro. The stability of the Pab1p–Arf1p complex depends on the presence of mRNA. Both symmetrically distributed mRNAs as well as the asymmetrically localized ASH1 mRNA are found in association with Arf1p. Remarkably, Arf1p and Pab1p are both required to restrict ASH1 mRNA to the bud tip. Arf1p and coatomer play an unexpected role in localizing mRNA independent and downstream of the SHE machinery. Hereby acts the SHE machinery in long-range mRNA transport, whereas COPI vesicles could act as short-range and localization vehicles. The endoplasmic reticulum (ER)–Golgi shuttle might be involved in concentrating mRNA at the ER.

scholarly journals Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation

2016 ◽  
Vol 113 (28) ◽  
pp. E4015-E4024 ◽  
Author(s):  
Yi-Chang Liu ◽  
Danica Galonić Fujimori ◽  
Jonathan S. Weissman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Disulfide Isomerase ◽  
Misfolded Proteins ◽  
Disulfide Isomerase ◽  
Licensing Factor ◽  
Folded Proteins ◽  
Protein Disulfide

Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

scholarly journals Cholera Toxin Is Exported from Microsomes by the Sec61p Complex

2000 ◽  
Vol 148 (6) ◽  
pp. 1203-1212 ◽  
Author(s):  
Anton Schmitz ◽  
Helga Herrgen ◽  
Alexandra Winkeler ◽  
Volker Herzog
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
De Novo ◽  
Bacterial Toxins ◽  
Protein Export ◽  
In Vitro Translation ◽  
Unknown Mechanism ◽  
A Cell ◽  
Er Export

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate–dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.

scholarly journals Structure–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES

2017 ◽  
Vol 217 (3) ◽  
pp. 959-974 ◽  
Author(s):  
Shin Kawano ◽  
Yasushi Tamura ◽  
Rieko Kojima ◽  
Siqin Bala ◽  
Eri Asai ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Kluyveromyces Lactis ◽  
Lipid Transfer ◽  
Mitochondrial Membranes ◽  
Hydrophobic Pocket ◽  
X Ray ◽  
Lipid Exchange ◽  
Complex Functions

The endoplasmic reticulum (ER)–mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12–Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1–Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes.

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