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 Not just a cargo receptor for large cargoes; an emerging role of TANGO1 as an organizer of ER exit sites

2019 ◽  
Vol 166 (2) ◽  
pp. 115-119 ◽  
Author(s):  
Kota Saito ◽  
Miharu Maeda
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Vesicle Formation ◽  
Wide Range ◽  
Copii Vesicle ◽  
Cargo Receptor ◽  
Er Exit Sites ◽  
Golgi Organization

Abstract Proteins synthesized within the endoplasmic reticulum (ER) are exported from ER exit sites via coat protein complex II (COPII)-coated vesicles. Although the mechanisms of COPII-vesicle formation at the ER exit sites are highly conserved among species, vertebrate cells secrete a wide range of materials, including collagens and chylomicrons, which form bulky structures within the ER that are too large to fit into conventional carriers. Transport ANd Golgi Organization 1 (TANGO1) was initially identified as a cargo receptor for collagens but has been recently rediscovered as an organizer of ER exit sites. We would like to review recent advances in the mechanism of large cargo secretion and organization of ER exit sites through the function of TANGO1.

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 Activation of the Mitogen-activated Protein Kinase, Slt2p, at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (10) ◽  
pp. 1772-1782 ◽  
Author(s):  
Xia Li ◽  
Yunrui Du ◽  
Steven Siegel ◽  
Susan Ferro-Novick ◽  
Peter Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Phosphatase ◽  
Normal Growth ◽  
Growth Conditions ◽  
Mitogen Activated Protein Kinase ◽  
Mitochondrial Inheritance ◽  
Daughter Cells ◽  
Late Step ◽  
Mitogen Activated Protein

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae . Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.

scholarly journals Loss of the Homotypic Fusion and Vacuole Protein Sorting or Golgi-Associated Retrograde Protein Vesicle Tethering Complexes Results in Gentamicin Sensitivity in the Yeast Saccharomyces cerevisiae

2006 ◽  
Vol 50 (2) ◽  
pp. 587-595 ◽  
Author(s):  
Mark C. Wagner ◽  
Elizabeth E. Molnar ◽  
Bruce A. Molitoris ◽  
Mark G. Goebl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intracellular Trafficking ◽  
Protein Sorting ◽  
Growth Conditions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vesicle Tethering ◽  
Yeast Deletion Library ◽  
Texas Red ◽  
Deletion Library ◽  
Logarithmic Growth

ABSTRACT Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.

scholarly journals Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (9) ◽  
pp. 1601-1615 ◽  
Author(s):  
Harsha Garadi Suresh ◽  
Aline Xavier da Silveira dos Santos ◽  
Wanda Kukulski ◽  
Jens Tyedmers ◽  
Howard Riezman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Metabolic Flux ◽  
Fatty Acid Synthetase ◽  
Growth Conditions ◽  
Lipid Homeostasis ◽  
Biosynthetic Enzymes ◽  
Glucose Depletion ◽  
Key Enzyme ◽  
Quantitative Changes

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

scholarly journals TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats

2016 ◽  
Vol 113 (36) ◽  
pp. 10061-10066 ◽  
Author(s):  
Wenfu Ma ◽  
Jonathan Goldberg
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Biochemical Analysis ◽  
Coat Proteins ◽  
Outer Coat ◽  
Receptor Molecule ◽  
X Ray ◽  
Er Exit Sites ◽  
Multiple Copies

The supramolecular cargo procollagen is loaded into coat protein complex II (COPII)-coated carriers at endoplasmic reticulum (ER) exit sites by the receptor molecule TANGO1/cTAGE5. Electron microscopy studies have identified a tubular carrier of suitable dimensions that is molded by a distinctive helical array of the COPII inner coat protein Sec23/24•Sar1; the helical arrangement is absent from canonical COPII-coated small vesicles. In this study, we combined X-ray crystallographic and biochemical analysis to characterize the association of TANGO1/cTAGE5 with COPII proteins. The affinity for Sec23 is concentrated in the proline-rich domains (PRDs) of TANGO1 and cTAGE5, but Sec23 recognizes merely a PPP motif. The PRDs contain repeated PPP motifs separated by proline-rich linkers, so a single TANGO1/cTAGE5 receptor can bind multiple copies of coat protein in a close-packed array. We propose that TANGO1/cTAGE5 promotes the accretion of inner coat proteins to the helical lattice. Furthermore, we show that PPP motifs in the outer coat protein Sec31 also bind to Sec23, suggesting that stepwise COPII coat assembly will ultimately displace TANGO1/cTAGE5 and compartmentalize its operation to the base of the growing COPII tubule.

scholarly journals Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI

2011 ◽  
Vol 194 (1) ◽  
pp. 61-75 ◽  
Author(s):  
Morihisa Fujita ◽  
Reika Watanabe ◽  
Nina Jaensch ◽  
Maria Romanova-Michaelides ◽  
Tadashi Satoh ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Posttranslational Modification ◽  
Protein Complex ◽  
Er Exit Sites ◽  
Ph Dependent ◽  
Copii Vesicles ◽  
Attachment Proteins ◽  
Acidic Compartments ◽  
P24 Proteins

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.

scholarly journals Requirements for Transitional Endoplasmic Reticulum Site Structure and Function in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (9) ◽  
pp. 1530-1545 ◽  
Author(s):  
Polina Shindiapina ◽  
Charles Barlowe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Structure And Function ◽  
Site Structure ◽  
Complex Ii ◽  
And Function ◽  
Coat Protein Complex

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-green fluorescent protein at tER sites was established to directly test requirements. tER sites remained active for ∼10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro, whereas depletion of phophatidylinositol selectively inhibited coat protein complex II (COPII) budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.

scholarly journals TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers

2018 ◽  
Vol 115 (52) ◽  
pp. E12255-E12264 ◽  
Author(s):  
Lin Yuan ◽  
Samuel J. Kenny ◽  
Juliet Hemmati ◽  
Ke Xu ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Coated Vesicles ◽  
Initiation Factor ◽  
Interacting Protein ◽  
Complex Ii ◽  
Transport Vesicles ◽  
Er Exit Sites ◽  
Copii Vesicles

Large coat protein complex II (COPII)-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER). The link between large cargo in the lumen of the ER and modulation of the COPII machinery remains unresolved. TANGO1 is required for PC secretion and interacts with PC and COPII on opposite sides of the ER membrane, but evidence suggests that TANGO1 is retained in the ER, and not included in normal size (<100 nm) COPII vesicles. Here we show that TANGO1 is exported out of the ER in large COPII-coated PC1 carriers, and retrieved back to the ER by the retrograde coat, COPI, mediated by the C-terminal RDEL retrieval sequence of HSP47. TANGO1 is known to target the COPII initiation factor SEC12 to ER exit sites through an interacting protein, cTAGE5. SEC12 is important for the growth of COPII vesicles, but it is not sorted into small budded vesicles. We found both cTAGE5 and SEC12 were exported with TANGO1 in large COPII carriers. In contrast to its exclusion from small transport vesicles, SEC12 was particularly enriched around ER membranes and large COPII carriers that contained PC1. We constructed a split GFP system to recapitulate the targeting of SEC12 to PC1 via the luminal domain of TANGO1. The minimal targeting system enriched SEC12 around PC1 and generated large PC1 carriers. We conclude that TANGO1, cTAGE5, and SEC12 are copacked with PC1 into COPII carriers to increase the size of COPII, thus ensuring the capture of large cargo.

Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae

2000 ◽  
Vol 381 (9-10) ◽  
pp. 1025-1028 ◽  
Author(s):  
Anke Prinz ◽  
Enno Hartmann ◽  
Kai-Uwe Kalies
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
Growth Conditions ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Tight Association ◽  
Biochemical Analyses

Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

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