Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion

Bas, Levent; Papinski, Daniel; Licheva, Mariya; Torggler, Raffaela; Rohringer, Sabrina; Schuschnig, Martina; Kraft, Claudine

doi:10.1083/jcb.201804028

Cited by 100 publications

(132 citation statements)

References 65 publications

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“…3E-G). These connections are in line with the pleiotropic effects observed for Ykt6 in diverse membrane-associated processes like ER-Golgi traffic (McNew et al, 1997;Fukasawa et al, 2004;Zhang & Hong, 2001), plasma membrane fusion (Gordon et al, 2017) and autophagy (Matsui et al, 2018;Takáts et al, 2018;Bas et al, 2018;Gao et al, 2018). We found nine candidates that were connected to endocytosis processes ( Fig.…”

Section: Ykt6 Acts At the Level Of Endosome Maturationsupporting

confidence: 87%

“…It has therefore been proposed to function as a membrane stress sensor within the secretory pathway of yeast (Dietrich et al, 2004). While a role for Ykt6 in homotypic fusion of ER and vacuolar membranes, retrograde Golgi trafficking in yeast and several steps of autophagosome formation under starvation conditions was demonstrated in human cells (Matsui et al, 2018), Drosophila fat body (Takáts et al, 2018) and yeast (Bas et al, 2018;Gao et al, 2018), its function in secretion in higher eukaryotes remains unclear. Considering the ability of Ykt6 to adapt to multiple cellular localizations, we here investigate Ykt6 as a candidate to orchestrate selected cargo sorting using the example of Wnt proteins.…”

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confidence: 99%

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Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Linnemannstöns

Witte

et al. 2018

Preprint

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Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion.However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6 RNAi-mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. We show that phosphomimicking Ykt6 accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies in a dose-dependent manner. Taken together, we show that exosomal sorting of Wnts is fine-tuned by a regulatory switch in Ykt6 shifting it from a membrane-bound to cytosolic state at the level of endosomal maturation.

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Section: Ykt6 Acts At the Level Of Endosome Maturationsupporting

confidence: 87%

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confidence: 99%

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Linnemannstöns

Witte

et al. 2018

Preprint

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“…In yeast, PI‐3‐P recruits a Rab7‐like GTPase, Ypt7 to the autophagosome, which in turn is necessary for recruitment of the Homotypic fusion and protein sorting (HOPS) complex. The HOPS complex allows for the assembly of SNARE proteins, which are important for the autophagosome–vacuole fusion process . This study also shows that presence of R‐SNARE, Ykt6, on the autophagosome and Q‐SNAREs Vam3, Vam7 and Vti1, on the vacuole are integral to this process .…”

Section: Roles Of Phosphoinositides In Autophagysupporting

confidence: 57%

Phosphoinositides in autophagy: current roles and future insights

2019

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Today, the importance of autophagy in physiological processes and pathological conditions is undeniable. Initially, autophagy merely was described as an evolutionarily conserved mechanism to maintain metabolic homeostasis in times of starvation; however, in recent years it is now apparent that autophagy is a powerful regulator of many facets of cellular metabolism, that its deregulation contributes to various human pathologies, including cancer and neurodegeneration, and that its modulation has considerable potential as a therapeutic approach. Different lipid species, including sphingolipids, sterols, and phospholipids, play important roles in the various steps of autophagy. In particular, there is accumulating evidence indicating the minor group of phospholipids called the phosphoinositides as key modulators of autophagy, including the signaling processes underlying autophagy initiation, autophagosome biogenesis and maturation. In this review, we discuss the known functions to date of the phosphoinositides in autophagy and attempt to summarize the kinases and phosphatases that regulate them as well as the proteins that bind to them throughout the autophagy program. We will also provide examples of how the control of phosphoinositides and their metabolizing enzymes is relevant to understanding many human diseases.Abbreviations ALR, autophagic lysosome reformation; CMT4J, Charcot-Marie-Tooth type 4J; ESI, electron spray ionization; FYCO1, FYVE and coiled-coiled domain-containing 1; GABARAP, gamma-aminobutyric acid receptor-associated protein; HOPS, homotypic fusion and protein sorting; HPLC, high performance liquid chromatography; KO, knockout; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MTM1, Myotubular myopathy 1; MTMRs, myotubularin family of phosphatases; mTORC1, mammalian target of rapamycin complex 1; OCRL, OculoCerebroRenal of Lowe; PDK1, phosphoinositide-dependent kinase-1; PH, pleckstrin homology; PI, phosphatidylinositol; PI-3,5-P 2 , phosphatidylinositol 3,5-bisphosphate; PI-3-P, phosphatidylinositol 3-monophosphate; PI-4,5-P 2 , phosphatidylinositol 4,5-bisphosphate; PI4P5K, phosphatidylinositol-4-phosphate 5-kinase; PI-5-P, phosphatidylinositol 5-monophosphate; PI5P4K, phosphatidylinositol-5-phosphate 4-kinase; PLCd1-PH, PH domain of phospholipase C d1; PLEKHM1, pleckstrin homology domain-containing protein family member 1; PM, plasma membrane; SynJ1, synaptojanin 1; TFEB, transcription factor EB; WIPI, WD-repeat protein interacting with phosphoinositides.

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“…In addition to its roles in the secretory and endocytic pathways, Ykt6 has recently been implicated in macroautophagy (hereafter referred to as autophagy) (Gao, Reggiori, & Ungermann, 2018) (Bas et al, 2018) (Takats et al, 2018) (Matsui et al, 2018). Autophagy is a highly conserved intracellular degradation pathway of long-lived proteins, misfolded proteins, and damaged organelles (Mizushima & Komatsu, 2011).…”

Section: Introductionmentioning

confidence: 99%

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

McGrath¹,

Dergai

Chung³

et al. 2020

Preprint

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1Membrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an 2 essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy 3 pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed 4 cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition 5 is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian 6 cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. 7The phosphorylation site we identified is highly conserved in evolution and is regulated by the 8 Ca 2+ -dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant 9 for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel 10 mechanism by which Ykt6 conformation and activity is regulated by Ca 2+ signaling with 11 implications in Parkinson's Disease in which Ykt6 has been shown to play a role.

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Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion

Abstract: Autophagosome fusion with vacuoles requires a conserved fusion machinery, though the topology remained unclear. Two papers in this issue, Bas et al. and Gao et al., uncover Ykt6 as the required autophagosomal SNARE.

Cited by 100 publications

References 65 publications

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Phosphoinositides in autophagy: current roles and future insights

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

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