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1.
J Cell Biol ; 223(4)2024 04 01.
Article in English | MEDLINE | ID: mdl-38319250

ABSTRACT

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.


Subject(s)
Endosomal Sorting Complexes Required for Transport , Endosomes , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Endocytosis , Endosomal Sorting Complexes Required for Transport/genetics , Endosomes/genetics , Lipids , Multivesicular Bodies , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Protein Transport
2.
Bio Protoc ; 12(9): e4403, 2022 May 05.
Article in English | MEDLINE | ID: mdl-35800464

ABSTRACT

Endosomal recycling is essential for the appropriate function of the endosome. During this process, endosomal coat complexes (i.e., retromer, and Mvp1) are recruited to the endosome, and deform its membrane to form recycling vesicles. To further analyze this, we developed a protocol for the immunoisolation of recycling vesicles from budding yeast. This method is a powerful way to characterize endosomal recycling pathways.

3.
Elife ; 112022 06 30.
Article in English | MEDLINE | ID: mdl-35770973

ABSTRACT

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY motif containing adaptor proteins. Several arrestin-related trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the plasma membrane recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the pombe orthologs of Rsp5 and Art1, Pub1, and Any1. Furthermore, we discover that the homologous to E6AP C-terminus (HECT) domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment, and activity of Rsp5 for the turnover of membrane proteins.


Subject(s)
Saccharomyces cerevisiae Proteins , Ubiquitin , Adaptor Proteins, Signal Transducing/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Nedd4 Ubiquitin Protein Ligases/metabolism , Poly(A)-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
4.
Sci Adv ; 8(13): eabm5149, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35363519

ABSTRACT

The general mechanisms by which ESCRTs (Endosomal Sorting Complexes Required for Transport) are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin are critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.

5.
J Cell Sci ; 135(5)2022 03 01.
Article in English | MEDLINE | ID: mdl-34415038

ABSTRACT

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.


Subject(s)
Carrier Proteins , Drosophila Proteins/genetics , Endoplasmic Reticulum , Vesicular Transport Proteins/genetics , Animals , Cell Membrane/metabolism , Drosophila melanogaster , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phosphatidylinositols , Saccharomyces cerevisiae
6.
Elife ; 102021 11 25.
Article in English | MEDLINE | ID: mdl-34821548

ABSTRACT

Protein glycosylation in the Golgi is a sequential process that requires proper distribution of transmembrane glycosyltransferase enzymes in the appropriate Golgi compartments. Some of the cytosolic machinery required for the steady-state localization of some Golgi enzymes are known but existing models do not explain how many of these enzymes are localized. Here, we uncover the role of an integral membrane protein in yeast, Erd1, as a key facilitator of Golgi glycosyltransferase recycling by directly interacting with both the Golgi enzymes and the cytosolic receptor, Vps74. Loss of Erd1 function results in mislocalization of Golgi enzymes to the vacuole/lysosome. We present evidence that Erd1 forms an integral part of the recycling machinery and ensures productive recycling of several early Golgi enzymes. Our work provides new insights on how the localization of Golgi glycosyltransferases is spatially and temporally regulated, and is finely tuned to the cues of Golgi maturation.


Subject(s)
Glycosyltransferases/metabolism , Membrane Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Glycosylation , Golgi Apparatus
7.
Elife ; 102021 09 15.
Article in English | MEDLINE | ID: mdl-34524084

ABSTRACT

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans.


Subject(s)
Endosomes/enzymology , GTP-Binding Proteins/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Sorting Nexins/metabolism , Vesicular Transport Proteins/metabolism , Endosomes/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation, Fungal , HeLa Cells , Humans , Protein Sorting Signals , Protein Transport , Proteolysis , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sorting Nexins/genetics , Time Factors , Ubiquitination , Vesicular Transport Proteins/genetics
8.
Elife ; 102021 05 24.
Article in English | MEDLINE | ID: mdl-34028356

ABSTRACT

ESCRT-III polymerization is required for all endosomal sorting complex required for transport (ESCRT)-dependent events in the cell. However, the relative contributions of the eight ESCRT-III subunits differ between each process. The minimal features of ESCRT-III proteins necessary for function and the role for the multiple ESCRT-III subunits remain unclear. To identify essential features of ESCRT-III subunits, we previously studied the polymerization mechanisms of two ESCRT-III subunits Snf7 and Vps24, identifying the association of the helix-4 region of Snf7 with the helix-1 region of Vps24 (Banjade et al., 2019a). Here, we find that mutations in the helix-1 region of another ESCRT-III subunit Vps2 can functionally replace Vps24 in Saccharomyces cerevisiae. Engineering and genetic selections revealed the required features of both subunits. Our data allow us to propose three minimal features required for ESCRT-III function - spiral formation, lateral association of the spirals through heteropolymerization, and binding to the AAA + ATPase Vps4 for dynamic remodeling.


Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression Regulation, Fungal , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity Relationship
9.
Trends Cell Biol ; 31(4): 269-283, 2021 04.
Article in English | MEDLINE | ID: mdl-33414051

ABSTRACT

Protein quality control (PQC) machineries play a critical role in selective identification and removal of mistargeted, misfolded, and aberrant proteins. This task is extremely complicated due to the enormous diversity of the proteome. It also requires nuanced and careful differentiation between 'normal' and 'folding intermediates' from 'abnormal' and 'misfolded' protein states. Multiple genetic and proteomic approaches have started to delineate the molecular underpinnings of how these machineries recognize their target and how their activity is regulated. In this review, we summarize our understanding of the various E3 ubiquitin ligases and associated machinery that mediate PQC in the endo-lysosome system in yeast and humans, how they are regulated, and mechanisms of target selection, with the intent of guiding future research in this area.


Subject(s)
Membrane Proteins , Proteomics , Humans , Lysosomes , Saccharomyces cerevisiae/genetics , Ubiquitination
10.
J Cell Biol ; 219(8)2020 08 03.
Article in English | MEDLINE | ID: mdl-32421152

ABSTRACT

Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.


Subject(s)
Calcineurin/metabolism , Endocytosis , Endosomal Sorting Complexes Required for Transport/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligase Complexes/metabolism , Calcineurin/genetics , Chitin Synthase/genetics , Chitin Synthase/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression Regulation, Fungal , Membrane Proteins/genetics , Protein Sorting Signals , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitination
11.
Mol Biol Cell ; 31(12): 1302-1313, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32267208

ABSTRACT

Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.


Subject(s)
Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Lipids/genetics , Membrane Proteins/metabolism , Mitochondrial Membranes/metabolism , Protein Transport/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
12.
Mol Biol Cell ; 30(25): 3057-3072, 2019 12 01.
Article in English | MEDLINE | ID: mdl-31618110

ABSTRACT

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins, which in the yeast Saccharomyces cerevisiae is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin--related trafficking (ART) adaptors. Here, we provide evidence that the ART protein family members are composed of an arrestin fold with interspersed disordered loops. Using Art1 as a model, we show that these loop and tail regions, while not strictly required for function, regulate its activity through two separate mechanisms. Disruption of one loop mediates Art1 substrate specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1's localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.


Subject(s)
DNA-Binding Proteins/metabolism , Endocytosis/physiology , Protein Transport/physiology , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Arrestins/metabolism , Cell Membrane/metabolism , DNA-Binding Proteins/physiology , Endosomal Sorting Complexes Required for Transport/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiology , Transcription Factors/physiology , Ubiquitin/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
13.
J Cell Biol ; 218(9): 2876-2886, 2019 09 02.
Article in English | MEDLINE | ID: mdl-31337624

ABSTRACT

Retromer is an evolutionarily conserved protein complex, which sorts functionally diverse membrane proteins into recycling tubules/vesicles from the endosome. Many of the identified cargos possess a recycling signal sequence defined as ØX[L/M/V], where Ø is F/Y/W. However, this sequence is present in almost all proteins encoded in the genome. Also, several identified recycling sequences do not follow this rule. How then does retromer precisely select its cargos? Here, we reveal that an additional motif is also required for cargo retrieval. The two distinct motifs form a bipartite recycling signal recognized by the retromer subunits, Vps26 and Vps35. Strikingly, Vps26 utilizes different binding sites depending on the cargo, allowing retromer to recycle different membrane proteins. Thus, retromer interacts with cargos in a more complex manner than previously thought, which facilitates precise cargo recognition.


Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vesicular Transport Proteins/metabolism , Binding Sites , Protein Transport/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Vesicular Transport Proteins/genetics
14.
Elife ; 82019 06 27.
Article in English | MEDLINE | ID: mdl-31246173

ABSTRACT

Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.


Subject(s)
Biopolymers/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Static Electricity , Amino Acid Sequence , Endosomal Sorting Complexes Required for Transport/chemistry , Endosomal Sorting Complexes Required for Transport/genetics , Mutation/genetics , Protein Binding , Protein Structure, Secondary , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Suppression, Genetic
15.
Methods Mol Biol ; 1998: 105-116, 2019.
Article in English | MEDLINE | ID: mdl-31250297

ABSTRACT

Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.


Subject(s)
Amino Acid Transport Systems, Basic/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Multivesicular Bodies/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Transport Systems, Basic/genetics , Endocytosis/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/isolation & purification , Green Fluorescent Proteins/chemistry , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Membranes, Artificial , Microscopy, Electron/methods , Microscopy, Fluorescence/methods , Mutation , Protein Multimerization/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Vacuoles/metabolism
16.
Methods Enzymol ; 619: 269-291, 2019.
Article in English | MEDLINE | ID: mdl-30910024

ABSTRACT

Covalent modification of proteins with ubiquitin dynamically regulates their function and fate. The ubiquitination of most plasma membrane proteins initiates endocytosis and ESCRT-mediated sorting to the lysosomal lumen for degradation. Powerful genetic approaches in the budding yeast Saccharomyces cerevisiae have been particularly instrumental in the discovery and elucidation of these molecular mechanisms, which are conserved in all eukaryotes. Here we provide two detailed protocols and tools for studying ubiquitination-dependent membrane trafficking mechanisms in yeast. The first utilizes fusions between a protein of interest and an auxotrophic marker to screen for mutants that affect ubiquitin-mediated endocytosis. The second method artificially ubiquitinates a protein of interest, allowing downstream trafficking steps to be studied independently from the regulatory signals that initiate endocytosis.


Subject(s)
Endocytosis , Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Protein Transport , Saccharomyces cerevisiae/cytology , Signal Transduction , Ubiquitination
17.
J Cell Biol ; 218(1): 234-250, 2019 01 07.
Article in English | MEDLINE | ID: mdl-30361468

ABSTRACT

Maintenance of organelle identity is profoundly dependent on the coordination between correct targeting of proteins and removal of mistargeted and damaged proteins. This task is mediated by organelle-specific protein quality control (QC) systems. In yeast, the endocytosis and QC of most plasma membrane (PM) proteins requires the Rsp5 ubiquitin ligase and ART adaptor network. We show that intracellular adaptors of Rsp5, Ear1, and Ssh4 mediate recognition and vacuolar degradation of PM proteins that escape or bypass PM QC systems. This second tier of surveillance helps to maintain cell integrity upon heat stress and protects from proteotoxicity. To understand the mechanism of the recognition of aberrant PM cargos by Ssh4-Rsp5, we mistarget multiple PM proteins de novo to the vacuolar membrane. We found that Ssh4-Rsp5 can target and ubiquitinate multiple lysines within a restricted distance from the membrane, providing a fail-safe mechanism for a diverse cargo repertoire. The mistargeting or misfolding of PM proteins likely exposes these lysines or shifts them into the "ubiquitination zone" accessible to the Ssh4-Rsp5 complex.


Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Cell Membrane/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression Regulation, Fungal , Intracellular Membranes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Ubiquitin-Protein Ligase Complexes/genetics , Actin-Related Protein 2-3 Complex , Adaptor Proteins, Vesicular Transport/metabolism , Amino Acid Sequence , Endocytosis , Endosomal Sorting Complexes Required for Transport/metabolism , Hot Temperature , Lysine/metabolism , Protein Transport , Proteolysis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Stress, Physiological , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitination , Vacuoles/metabolism
18.
Autophagy ; 14(9): 1654-1655, 2018.
Article in English | MEDLINE | ID: mdl-29995558

ABSTRACT

Membrane protein recycling is a fundamental process from yeast to humans. The lysosome (or vacuole in yeast) receives membrane proteins from the secretory, endocytic, and macroautophagy/autophagy pathways. Although some of these membrane proteins appear to be recycled, the molecular mechanisms underlying this retrograde trafficking are poorly understood. Our recent study revealed that the transmembrane autophagy protein Atg27 is recycled from the vacuole membrane using a 2-step recycling process. First, the Snx4 complex recycles Atg27 from the vacuole to the endosome. Then, the retromer complex mediates endosome-to-Golgi retrograde transport. Thus, 2 distinct protein complexes facilitate the sequential retrograde trafficking for Atg27. As far as we know, Atg27 is the first physiological substrate for the vacuole-to-endosome retrograde trafficking pathway.


Subject(s)
Intracellular Membranes/metabolism , Lysosomes/metabolism , Vacuoles/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolism , Membrane Fusion , Membrane Proteins/metabolism , Models, Biological , Protein Transport
19.
J Cell Biol ; 217(5): 1623-1632, 2018 05 07.
Article in English | MEDLINE | ID: mdl-29511122

ABSTRACT

The lysosome (or vacuole in yeast) is the central organelle responsible for cellular degradation and nutrient storage. Lysosomes receive cargo from the secretory, endocytic, and autophagy pathways. Many of these proteins and lipids are delivered to the lysosome membrane, and some are degraded in the lysosome lumen, whereas others appear to be recycled through unknown pathways. In this study, we identify the transmembrane autophagy protein Atg27 as a physiological cargo recycled from the vacuole. We reveal that Atg27 is delivered to the vacuole membrane and then recycled using a two-step recycling process. First, Atg27 is recycled from the vacuole to the endosome via the Snx4 complex and then from the endosome to the Golgi via the retromer complex. During the process of vacuole-to-endosome retrograde trafficking, Snx4 complexes assemble on the vacuolar surface and recognize specific residues in the cytoplasmic tail of Atg27. This novel pathway maintains the normal composition and function of the vacuole membrane.


Subject(s)
Endocytosis , Intracellular Membranes/metabolism , Lysosomes/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vacuoles/metabolism , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/metabolism , Biological Transport , Endosomes/metabolism , Mutation/genetics , Phosphatidylinositol Phosphates/metabolism , Protein Domains , Saccharomyces cerevisiae Proteins/chemistry
20.
Elife ; 62017 06 29.
Article in English | MEDLINE | ID: mdl-28661397

ABSTRACT

The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.


Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomes/metabolism , Ubiquitination , Down-Regulation , Endosomal Sorting Complexes Required for Transport/genetics , Genetic Testing , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
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