STX19 is an unusual Qa-SNARE as it lacks a C-terminal transmembrane domain. However, it is efficiently targeted to post-Golgi membranes. Here, we set out to determine the intracellular localisation of endogenous STX19 and elucidate the mechanism by which it is targeted to membranes. We have found that a pool of STX19 is localised to tubular recycling endosomes where it colocalises with MICAL-L1 and Rab8 (which has Rab8a and Rab8b forms). Using a combination of genetic, biochemical and cell-based approaches, we have identified that STX19 is S-acylated at its C-terminus and is a substrate for several Golgi-localised S-acyltransferases, suggesting that STX19 is initially S-acylated at the Golgi before trafficking to the plasma membrane and endosomes. Surprisingly, we have found that S-acylation is a key determinant in targeting STX19 to tubular recycling endosomes, suggesting that S-acylation may play a general role in directing proteins to this compartment. In addition, S-acylation also protects STX19 from proteosomal degradation, indicating that S-acylation regulates the function of STX19 at multiple levels.This article has an associated First Person interview with the first author of the paper.
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.