Apg13p and Vac8p Are Part of a Complex of Phosphoproteins That Are Required for Cytoplasm to Vacuole Targeting

Scott, Sidney V.; Nice, Daniel C.; Nau, Johnathan J.; Weisman, Lois S.; Kamada, Yoshiaki; Keizer‐Gunnink, Ineke; Funakoshi, Tomoko; Veenhuis, Marten; Ohsumi, Yoshinori; Klionsky, Daniel J.

doi:10.1074/jbc.m002813200

Cited by 219 publications

(289 citation statements)

References 43 publications

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“…To simplify our analysis, we took advantage of the Cvt pathway phenotype of the vac8D background to monitor selective autophagy; in the vac8D strain only prApe1 accumulates in rich conditions, but there is rapid vacuolar delivery following the induction of autophagy. 41 Thus, a shift from rich to starvation conditions essentially corresponds to a pulsechase analysis, and prevents the complicating accumulation of mature Ape1 that would otherwise be present. In the atg9D vac8D strain only prApe1 could be detected even after the induction of autophagy (Fig.…”

Section: Phosphorylation Of Atg9 S122 Is Important For Selective Automentioning

confidence: 99%

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Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

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Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.

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Section: Phosphorylation Of Atg9 S122 Is Important For Selective Automentioning

confidence: 99%

“…Gene deletions or integrations were performed using a standard method. 41 Cells were cultured in rich medium (YPD; 1% …”

Section: Strains Media and Growth Conditionsmentioning

confidence: 99%

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

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“…Two members of the vacuole-specific Myo2p transport complex, Myo2p and Vac8p, have been shown to be phosphorylated (Scott et al, 2000;Legesse-Miller et al, 2006), and more recently we found that Vac17p is also phosphorylated (Peng and Weisman, 2008). We tested the phosphorylation status of all three proteins.…”

Section: Ptc1 Is Required For the Proper Association Of The Vacuole Tmentioning

confidence: 99%

PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

Jin

Eves

Tang

et al. 2009

MBoC

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Organelle inheritance occurs during cell division. In Saccharomyces cerevisiae, inheritance of the vacuole, and the distribution of mitochondria and cortical endoplasmic reticulum are regulated by Ptc1p, a type 2C protein phosphatase. Here we show that PTC1/VAC10 controls the distribution of additional cargoes moved by a myosin-V motor. These include peroxisomes, secretory vesicles, cargoes of Myo2p, and ASH1 mRNA, a cargo of Myo4p. We find that Ptc1p is required for the proper distribution of both Myo2p and Myo4p. Surprisingly, PTC1 is also required to maintain the steady-state levels of organelle-specific receptors, including Vac17p, Inp2p, and Mmr1p, which attach Myo2p to the vacuole, peroxisomes, and mitochondria, respectively. Furthermore, Vac17p fused to the cargo-binding domain of Myo2p suppressed the vacuole inheritance defect in ptc1⌬ cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components. INTRODUCTIONDuring each cell cycle, cytoplasmic organelles are actively distributed between dividing cells to maintain organelle copy number and volume. The yeast Saccharomyces cerevisiae is an excellent model system for studying the spatial and temporal control of organelle inheritance. In yeast, several organelles are transmitted from mother to daughter cells. These include the vacuole, mitochondria, the endoplasmic reticulum (ER), late-Golgi elements and peroxisomes (reviewed in Weisman, 2006).Early in the cell cycle, a portion of each organelle is transported into the emerging bud. The polarized transport of most organelles from the mother to the bud is an active process that requires the actin cytoskeleton, myosin-V motors, and receptor proteins, which physically connect the motor to organelle cargoes (Beach et al., 2000;Yin et al., 2000;Boldogh et al., 2001;Barr, 2002;Bretscher, 2003;Du et al., 2004;Fagarasanu et al., 2006bFagarasanu et al., , 2007Weisman, 2006). Thus, formation of a complex between the motor and receptor protein is important for polarized organelle transport.Similar to yeast, in vertebrates, myosin-V motors move cargoes along the actin cytoskeleton. The best studied cargo of vertebrate myosin-V are melanosomes, which are moved in melanocytes by myosin-Va. Melanosomes attach to myosin-Va through Rab27a and melanophilin (Fukuda and Kuroda, 2002;Wu et al., 2002). Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V-based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V ( Kinkema and Schiefelbein, 1994;Provance and Mercer, 1999;Tuma and Gelfand, 1999;Desnos et al., 2007;Li and Nebenfuhr, 2007;Sheets et al., 2007;Shimmen, 2007).Ge...

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“…Despite their differences, these pathways share a number of common molecular events. For example, Atg7/Gsa7/Apg7, Atg1/Gsa10/Apg1/Aut3, Atg2/Gsa11/Apg2, Atg18/Gsa12/Cvt18, and Vps15 are required for pexophagy, autophagy, and CVT pathways, whereas Atg11/Gsa9/Cvt9 and Vac8 are essential for pexophagy and CVT pathways, but not autophagy (Stasyk et al, 1999;Yuan et al, 1999;Scott et al, 2000;Guan et al, 2001;Kim et al, 2001;Strømhaug and Klionsky, 2001;Wang et al, 2001;Huang and Klionsky, 2002;Abeliovich et al, 2003). Because many autophagy genes and their orthologues have been identified using different yeast models, the nomenclature for these genes had become confusing.…”

Section: Introductionmentioning

confidence: 99%

PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy inPichia pastoris

Chang

Schröder

Thomson

et al. 2005

MBoC

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When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.

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Apg13p and Vac8p Are Part of a Complex of Phosphoproteins That Are Required for Cytoplasm to Vacuole Targeting

Cited by 219 publications

References 43 publications

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy inPichia pastoris

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