Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity

Abstract

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.

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

“…S8 B , step two). Supporting our findings, a phosphorylation driven membrane recruitment for Ykt6 was also reported while this paper was under review ( 43 ). The conformational mechanism of activation might not be restricted to Ykt6 since the S174 phosphorylation site is also evolutionarily conserved across other R-SNARE proteins ( 44 ).…”

Different conformational dynamics of SNARE protein Ykt6 among yeast and mammals

“…In other words, phosphorylation of Ser174/Ser177 regulates both conformational state of Ykt6 and its protein binding property in mammals and C. elegans , but phosphorylation only affects the protein binding capacity of Ykt6 in yeast. Besides Ser174, several other phosphorylation sites on Ykt6 have been identified ( 15 , 16 ), we anticipate that more regulatory mechanisms and delicate differences across species are yet to be discovered.…”

“…The conformational state of the longin SNARE Ykt6 is tightly related to its cellular localization and function. It has been revealed that the conformational state could be regulated by the lipid modification ( 11 , 22 ) and phosphorylation ( 15 , 16 , 17 ). However, it is intriguing that the function and conformational regulation of Ykt6 exhibit differences among various species ( 17 , 18 , 19 , 20 , 23 ).…”

“…The structure of r Ykt6/DPC complex (Protein Data Bank-ID: 3KYQ) is considered to mimic the farnesylated closed form of Ykt6 ( 13 ). Besides the lipidation, it was recently found that the conformation of Ykt6 could also be regulated by phosphorylation ( 15 , 16 , 17 ). Phosphorylation at the evolutionarily conserved site S174 in human Ykt6 ( h Ykt6) drives the conversion from a closed cytosolic to an open membrane-bound state ( 17 ).…”