scholarly journals Vacuolar protein Tag1 and Atg1–Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae

2021 ◽  
Vol 134 (4) ◽  
pp. jcs253682
Author(s):  
Shintaro Kira ◽  
Masafumi Noguchi ◽  
Yasuhiro Araki ◽  
Yu Oikawa ◽  
Tamotsu Yoshimori ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Membrane Protein ◽  
Nitrogen Starvation ◽  
Genetic Screen ◽  
Vacuolar Membrane ◽  
Phagophore Assembly Site ◽  
Vacuolar Protein ◽  
Assembly Site ◽  
Starvation Conditions

ABSTRACTUnder starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10–12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

scholarly journals Dual Sorting of the Saccharomyces cerevisiae Vacuolar Protein Sna4p

2009 ◽  
Vol 8 (3) ◽  
pp. 278-286 ◽  
Author(s):  
W. Pokrzywa ◽  
B. Guerriat ◽  
J. Dodzian ◽  
P. Morsomme
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Membrane Protein ◽  
Ubiquitin Ligase ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Small Family ◽  
Vacuolar Protein

ABSTRACT Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plants and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVBs). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic dileucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway by using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine, and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localize to both the vacuolar membrane and the lumen.

scholarly journals Self-Interaction Is Critical for Atg9 Transport and Function at the Phagophore Assembly Site during Autophagy

2008 ◽  
Vol 19 (12) ◽  
pp. 5506-5516 ◽  
Author(s):  
Congcong He ◽  
Misuzu Baba ◽  
Yang Cao ◽  
Daniel J. Klionsky
Keyword(s):  
Membrane Protein ◽  
Membrane Transport ◽  
Integral Membrane Protein ◽  
Human Diseases ◽  
Anterograde Transport ◽  
Membrane Flow ◽  
Phagophore Assembly Site ◽  
And Function ◽  
Assembly Site ◽  
Starvation Conditions

Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and is proposed to play a key role in membrane transport; the phagophore presumably expands by vesicular addition to form a complete autophagosome. It is not clear through what mechanism Atg9 functions at the phagophore assembly site (PAS). Here we report that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses reveal that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Our results support a model in which Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation.

scholarly journals Retrieval of Resident Late-Golgi Membrane Proteins from the Prevacuolar Compartment of Saccharomyces cerevisiae Is Dependent on the Function of Grd19p

1998 ◽  
Vol 140 (3) ◽  
pp. 577-590 ◽  
Author(s):  
Wolfgang Voos ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Localization ◽  
Transport Processes ◽  
Carboxypeptidase Y ◽  
Golgi Membrane ◽  
Cytosolic Domain ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

The dynamic vesicle transport processes at the late-Golgi compartment of Saccharomyces cerevisiae (TGN) require dedicated mechanisms for correct localization of resident membrane proteins. In this study, we report the identification of a new gene, GRD19, involved in the localization of the model late-Golgi membrane protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and lumenal domains of the alkaline phosphatase [ALP]), which localizes to the yeast TGN. A grd19 null mutation causes rapid mislocalization of the late-Golgi membrane proteins A-ALP and Kex2p to the vacuole. In contrast to previously identified genes involved in late-Golgi membrane protein localization, grd19 mutations cause only minor effects on vacuolar protein sorting. The recycling of the carboxypeptidase Y sorting receptor, Vps10p, between the TGN and the prevacuolar compartment is largely unaffected in grd19Δ cells. Kinetic assays of A-ALP trafficking indicate that GRD19 is involved in the process of retrieval of A-ALP from the prevacuolar compartment. GRD19 encodes a small hydrophilic protein with a predominantly cytosolic distribution. In a yeast mutant that accumulates an exaggerated form of the prevacuolar compartment (vps27), Grd19p was observed to localize to this compartment. Using an in vitro binding assay, Grd19p was found to interact physically with the cytosolic domain of DPAP A. We conclude that Grd19p is a component of the retrieval machinery that functions by direct interaction with the cytosolic tails of certain TGN membrane proteins during the sorting/budding process at the prevacuolar compartment.

Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A

1993 ◽  
Vol 13 (9) ◽  
pp. 5567-5581
Author(s):  
M J Blacketer ◽  
C M Koehler ◽  
S G Coats ◽  
A M Myers ◽  
P Madaule
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Nitrogen Starvation ◽  
Gene Dosage ◽  
Protein Phosphatase 2A ◽  
Pseudohyphal Growth ◽  
Phosphatase 2A ◽  
Pseudohyphal Differentiation ◽  
Novel Protein

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.

scholarly journals Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.

1993 ◽  
Vol 13 (9) ◽  
pp. 5567-5581 ◽  
Author(s):  
M J Blacketer ◽  
C M Koehler ◽  
S G Coats ◽  
A M Myers ◽  
P Madaule
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Nitrogen Starvation ◽  
Gene Dosage ◽  
Protein Phosphatase 2A ◽  
Pseudohyphal Growth ◽  
Phosphatase 2A ◽  
Pseudohyphal Differentiation ◽  
Novel Protein

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.

scholarly journals Spatial control of avidity regulates initiation and progression of selective autophagy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David M. Hollenstein ◽  
Mariya Licheva ◽  
Nicole Konradi ◽  
David Schweida ◽  
Hector Mancilla ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Vacuolar Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Autophagosome Formation ◽  
Spatial Control ◽  
Selective Autophagy ◽  
Phagophore Assembly Site ◽  
Assembly Site ◽  
Phosphatidylinositol 3

AbstractAutophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

Cdc1 and the Vacuole Coordinately Regulate Mn2+ Homeostasis in the Yeast Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1787-1798 ◽  
Author(s):  
Madan Paidhungat ◽  
Stephen Garrett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Double Mutant ◽  
Vacuolar Membrane ◽  
Deletion Strain ◽  
Growth Defect ◽  
Yeast Saccharomyces Cerevisiae ◽  
Suppressor Genes ◽  
Essential Protein ◽  
Essential Function

Abstract The yeast CDC1 gene encodes an essential protein that has been implicated in the regulation of cytosolic [Mn2+]. To identify factors that impinge upon Cdc1 or the Cdc1-dependent process, we isolated secondsite suppressors of the conditional cdc1-1(Ts) growth defect. Recessive suppressors define 15 COS (CdcOne Suppressor) genes. Seven of the fifteen COS genes are required for biogenesis of the vacuole, an organelle known to sequester intracellular Mn2+. An eighth gene, COS16, encodes a vacuolar membrane protein that seems to be involved in Mn2+ homeostasis. These results suggest mutations that block vacuolar Mn2+ sequestration compensate for defects in Cdc1 function. Interestingly, Cdc1 is dispensable in a cos16Δ deletion strain, and a cdc1Δ cos16Δ double mutant exhibits robust growth on medium supplemented with Mn2+. Thus, the single, essential function of Cdc1 is to regulate intracellular, probably cytosolic, Mn2+.

scholarly journals Hansenula Polymorpha Vac8: A Vacuolar Membrane Protein Required for Vacuole Inheritance and Nucleus-Vacuole Junction Formation

Contact ◽  
2020 ◽  
Vol 3 ◽  
pp. 251525642097492
Author(s):  
Ritika Singh ◽  
Justyna Wróblewska ◽  
Rinse de Boer ◽  
Ida J. van der Klei
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Hansenula Polymorpha ◽  
Vacuolar Membrane ◽  
Peroxisome Biogenesis ◽  
Contact Sites ◽  
Vacuole Fusion ◽  
Junction Formation ◽  
Fusion Nucleus ◽  
Vacuole Inheritance

Saccharomyces cerevisiae Vac8 is a vacuolar membrane protein, which functions in vacuole inheritance and fusion, nucleus-vacuole junctions, autophagy and the cytoplasm-to-vacuole-targeting pathway. Here, we analyzed Vac8 of the yeast Hansenula polymorpha. We show that HpVac8 localizes to the vacuolar membrane and concentrates in patches at nucleus-vacuole junctions. Analysis of a VAC8 deletion strain indicated that HpVac8 is required for vacuole inheritance and the formation of nuclear-vacuole junctions, but not for vacuole fusion. Previously, organelle proteomics resulted in the identification of Vac8 in peroxisomal fractions isolated from H. polymorpha and S. cerevisiae. However, deletion of H. polymorpha VAC8 had no effect on peroxisome biogenesis or peroxisome-vacuole contact sites.

γ-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione

2001 ◽  
Vol 359 (3) ◽  
pp. 631-637 ◽  
Author(s):  
Karim MEHDI ◽  
Jacques THIERIE ◽  
Michel J. PENNINCKX
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Starvation ◽  
Coupled System ◽  
Vacuolar Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Strains ◽  
Gsh Metabolism ◽  
Glutamyl Transpeptidase ◽  
Hydrolysis Of ◽  
Wild Type Yeast

In the yeast Saccharomyces cerevisiae, the enzyme γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Σ1278b, under conditions of γ-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of γ-GT displayed a Km (app) of 18±2mM and a Vmax (app) of 48.5±5nmol of GSH/min per mg of protein. Vacuoles containing γ-GT displayed practically the same Km, but a higher Vmax (app) (150±12nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking γ-GT showed no increase in Vmax (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H+) ATPase-coupled system. The Vmax (app)-increasing effect of γ-GT concerns only the YCF1 system. γ-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing γ-GT was also measured. It is proposed that γ-GT and a l-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids l-glutamate, l-cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying γ-GT physiology and GSH metabolism.

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