Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex

Abstract

The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requires SNARE proteins, the homotypic vacuole fusion and protein sorting ( HOPS ) tethering complex, the RAB 7 GTP ase Ypt7, and its guanine nucleotide exchange factor ( GEF ) Mon1‐Ccz1. We and others recently identified Ykt6 as the autophagosomal SNARE protein. However, it has not been resolved when and how lipid‐anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum ( ER )‐resident Dsl1 complex and COPII ‐coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event.

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity

“…However, in Ykt6-3E overexpressing cells, those endosomes were significantly smaller than in Ykt6-WT ( Figure 5 B), indicating that the mutated SNARE domain might hinder endosomal fusion events activated by Rab5Q79L. Interestingly, under serum-fed conditions, Ykt6 had no effect on the levels of p62, as a readout of autophagy induction ( Figure 5 C), suggesting that the non-canonical role of Ykt6 in autophagy could be starvation-dependent [ 51 , 52 ].…”

“…Under normal growth conditions, these processes seem to be more important for cellular function, than its role in autophagosome formation. As it has also been reported that Ykt6 is involved in autophagosome–lysosome fusion in human cells and Drosophila fat body under starvation-induced conditions [ 15 , 19 ], it is possible that Ykt6 is allocated to alternate compartments, especially under nutrient stress condition [ 51 , 52 ].…”

“…Likely, an additional step is required to render Ykt6 fusion-competent, as Ykt6-3E did not rescue Ykt6 KD in vivo , but it had no dominant-negative effects in the presence of endogenous Ykt6. This last step toward fusion-competent Ykt6 could be achieved by dephosphorylation [ 52 ], for example by calcineurin, as suggested by a recent study [ 54 ].…”

A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6

“…Stx17 has been shown to play a role during autophagy initiation by assembling protein complexes necessary for the preautophagosomal structure (mPAS) ( 46 ). Moreover, it was recently shown that in yeast, phosphorylation of Ykt6 by Atg1 kinase also plays an inhibitory role during autophagosome formation, and dephosphorylation of Ykt6, by an unknown phosphatase, is necessary to complete autophagosome formation ( 47 ). Our data fit with these observations and suggest a model in which the Ca 2+ -CaN cascade posttranslationally regulates the early phases of autophagy through regulation of Ykt6 activity.…”

Phosphoregulation of the autophagy machinery by kinases and phosphatases

“…Fusion of autophagosomes with the lytic compartment requires the R-SNARE Ykt6 in both yeast and mammals [179][180][181]. Ykt6 is directly phosphorylated at Ser182 and Ser1832 by Atg1, and another Ytk6 phosphorylation site was identified at Thr158 (Figure 2, Table 1) [53,182,183]. Phosphorylation of these sites inhibits autophagosome-vacuole fusion by preventing Ykt6 bundling with the vacuolar Q-SNAREs Vam3 and Vti1 [182,183].…”