Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion

The endoplasmic reticulum (ER)–Golgi-independent, unconventional secretion of Acb1 requires many different proteins. They include proteins necessary for the formation of autophagosomes, proteins necessary for the fusion of membranes with the endosomes, proteins of the multivesicular body pathway, and the cell surface target membrane SNARE Sso1, thereby raising the question of what achieves the connection between these diverse proteins and Acb1 secretion. In the present study, we now report that, upon starvation in Saccharomyces cerevisiae, Grh1 is collected into unique membrane structures near Sec13-containing ER exit sites. Phosphatidylinositol 3 phosphate, the ESCRT (endosomal sorting complex required for transport) protein Vps23, and the autophagy-related proteins Atg8 and Atg9 are recruited to these Grh1-containing membranes, which lack components of the Golgi apparatus and the endosomes, and which we call a novel compartment for unconventional protein secretion (CUPS). We describe the cellular proteins required for the biogenesis of CUPS, which we believe is the sorting station for Acb1’s release from the cells.

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Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata

BMC Biology ◽

10.1186/s12915-021-01077-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Shweta V. Pipaliya ◽

Rui Santos ◽

Dayana Salas-Leiva ◽

Erina A. Balmer ◽

Corina D. Wirdnam ◽

...

Keyword(s):

Evolutionary Dynamics ◽

Gastrointestinal Disease ◽

Multivesicular Body ◽

Giardia Intestinalis ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Parasitic Lifestyle ◽

Cellular Compartments ◽

Related Proteins ◽

First Time

Abstract Background Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia’s pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. Results We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. Conclusions Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

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Unexpected organellar locations of ESCRT machinery in Giardia intestinalis and complex evolutionary dynamics spanning the transition to parasitism in the lineage Fornicata

10.1101/2021.02.11.430673 ◽

2021 ◽

Author(s):

Shweta V. Pipaliya ◽

Rui Santos ◽

Dayana Salas-Leiva ◽

Erina A. Balmer ◽

Corina D. Wirdnam ◽

...

Keyword(s):

Evolutionary Dynamics ◽

Gastrointestinal Disease ◽

Multivesicular Body ◽

Giardia Intestinalis ◽

Endosomal Sorting ◽

Escrt Machinery ◽

Parasitic Lifestyle ◽

Cellular Compartments ◽

Related Proteins ◽

First Time

ABSTRACTComparing a parasitic lineage to its free-living relatives is a powerful way to understand how the evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia’s pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species.We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the Endoplasmic Reticulum, and for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia, and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, and associating to multiple cellular locations and presenting changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

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Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽

10.1186/s12915-021-01048-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Cheng-Wen He ◽

Xue-Fei Cui ◽

Shao-Jie Ma ◽

Qin Xu ◽

Yan-Peng Ran ◽

...

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Stationary Phase ◽

Molecular Mechanisms ◽

Multivesicular Body ◽

Degradation Process ◽

Vacuolar Membrane ◽

Yeast Cells ◽

Membrane Structures ◽

Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

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Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

The Journal of Cell Biology ◽

10.1083/jcb.200312034 ◽

2004 ◽

Vol 165 (1) ◽

pp. 111-122 ◽

Cited By ~ 398

Author(s):

Matthew N.J. Seaman

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Multivesicular Body ◽

Carboxypeptidase Y ◽

Reporter Protein ◽

Rapid Degradation ◽

Endosomal Sorting ◽

Lysosome Biogenesis ◽

Golgi Transport ◽

Efficient Retrieval

fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called “retromer” that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

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VPS37A directs ESCRT recruitment for phagophore closure

The Journal of Cell Biology ◽

10.1083/jcb.201902170 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3336-3354 ◽

Cited By ~ 17

Author(s):

Yoshinori Takahashi ◽

Xinwen Liang ◽

Tatsuya Hattori ◽

Zhenyuan Tang ◽

Haiyan He ◽

...

Keyword(s):

Mammalian Cells ◽

Growth Factor Receptor ◽

Multivesicular Body ◽

Regulatory Mechanisms ◽

Endosomal Sorting ◽

Aaa Atpase ◽

Escrt Machinery ◽

Genome Wide ◽

A Genome ◽

Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718921115 ◽

2018 ◽

Vol 115 (19) ◽

pp. E4396-E4405 ◽

Cited By ~ 43

Author(s):

Sebastian Bänfer ◽

Dominik Schneider ◽

Jenny Dewes ◽

Maximilian T. Strauss ◽

Sven-A. Freibert ◽

...

Keyword(s):

Secretory Pathway ◽

Direct Interaction ◽

Multivesicular Body ◽

Dominant Negative ◽

Amino Terminus ◽

Multivesicular Bodies ◽

Dramatic Decrease ◽

Endosomal Sorting ◽

Galectin 3 ◽

Binding Lectin

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

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A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805504115 ◽

2018 ◽

Vol 115 (38) ◽

pp. E8900-E8908 ◽

Cited By ~ 19

Author(s):

Jessica B. A. Sadler ◽

Dawn M. Wenzel ◽

Lauren K. Williams ◽

Marta Guindo-Martínez ◽

Steven L. Alam ◽

...

Keyword(s):

Cancer Susceptibility ◽

Genome Instability ◽

Multivesicular Body ◽

Mitotic Checkpoint ◽

Body Protein ◽

Endosomal Sorting ◽

Daughter Cells ◽

Dna Replication Stress ◽

Human Polymorphism ◽

Mitotic Stress

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4CT232) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

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VPS4 triggers constriction and cleavage of ESCRT-III helical filaments

Science Advances ◽

10.1126/sciadv.aau7198 ◽

2019 ◽

Vol 5 (4) ◽

pp. eaau7198 ◽

Cited By ~ 26

Author(s):

Sourav Maity ◽

Christophe Caillat ◽

Nolwenn Miguet ◽

Guidenn Sulbaran ◽

Gregory Effantin ◽

...

Keyword(s):

High Speed ◽

Membrane Structures ◽

Vesicle Budding ◽

Endosomal Sorting ◽

Force Microscopy ◽

Cellular Processes ◽

Membrane Fission ◽

End Caps

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, we show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Our results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. We propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.

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The ESCRT pathway and HIV-1 budding

Biochemical Society Transactions ◽

10.1042/bst0370181 ◽

2009 ◽

Vol 37 (1) ◽

pp. 181-184 ◽

Cited By ~ 75

Author(s):

Yoshiko Usami ◽

Sergei Popov ◽

Elena Popova ◽

Michio Inoue ◽

Winfried Weissenhorn ◽

...

Keyword(s):

Susceptibility Gene ◽

Multivesicular Body ◽

Intramolecular Interactions ◽

Transport Pathway ◽

Interacting Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Binding Partner ◽

Protein X ◽

Hiv 1

HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPXnL (Leu-Tyr-Pro-Xaan-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

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ESCRT-III on endosomes: new functions, new activation pathway

Biochemical Journal ◽

10.1042/bj20151115 ◽

2016 ◽

Vol 473 (2) ◽

pp. e5-e8 ◽

Cited By ~ 5

Author(s):

Philip Woodman

Keyword(s):

Mhc Class I ◽

Protein Tyrosine Phosphatase ◽

Protein Complexes ◽

Tyrosine Phosphatase ◽

Multivesicular Body ◽

Class I ◽

Recent Issue ◽

Endosomal Sorting ◽

Histocompatibility Complex ◽

Biochemical Journal

The multivesicular body (MVB) pathway sorts ubiquitinated membrane cargo to intraluminal vesicles (ILVs) within the endosome, en route to the lysosomal lumen. The pathway involves the sequential action of conserved protein complexes [endosomal sorting complexes required for transport (ESCRTs)], culminating in the activation by ESCRT-II of ESCRT-III, a membrane-sculpting complex. Although this linear pathway of ESCRT activation is widely accepted, a study by Luzio and colleagues in a recent issue of the Biochemical Journal suggests that there is greater complexity in ESCRT-III activation, at least for some MVB cargoes. They show that ubiquitin-dependent sorting of major histocompatibility complex (MHC) class I to the MVB requires the central ESCRT-III complex but does not involve either ESCRT-II or functional links between ESCRT-II and ESCRT-III. Instead, they propose that MHC class I utilizes histidine-domain protein tyrosine phosphatase (HD-PTP), a non-canonical ESCRT interactor, to promote ESCRT-III activation.

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