Snazarus and its human ortholog SNX25 regulate autophagic flux by affecting VAMP8 endocytosis

Abstract

Autophagy, the degradation and recycling of cytosolic components in the lysosome, is an essential cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs can impact autophagy. To gain a better understanding of their functions in vivo under nutrient starvation, we screened all Drosophila SNXs by RNAi in the fat body. Significantly, depletion of snazarus (snz) strongly impacted autolysosome formation and led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with snz depletion and snz roles were conserved in human cells. SNX25 is the closest ortholog to snz, and we demonstrate a role for it in VAMP8 trafficking. We found that this activity was dependent on the SNX25 PX domain, and independent of SNX25 anchoring at the ER. We also demonstrate that differentially spliced forms of SNX14 and SNX25 are present in cancer cells. This work identifies a conserved role for snz/SNX25 as regulators of autophagic flux, and show differential isoform expression between orthologs.

Structural predictions of the SNX-RGS proteins suggest they belong to a new class of lipid transfer proteins

“…Relative to this 'core' structure the N-terminal membrane anchor, and intervening RGS and PX domains adopt diverse orientations pointing to a substantial degree of structural dynamics in the full-length proteins. Note that the human SNX25 sequence in the AlphaFold2 database (Uniprot ID Q9H3E2) lacks the N-terminal membrane anchor as previously identified 5,28 (Uniprot A0A494C0S0). We also performed predictions of the full SNX25 sequence using the online ColabFold pipeline 29 , which show a similar structure but with the additional Nterminal -helical membrane (Fig.…”

“…The structural predictions of the full-length proteins from different species including S. cerevisiae, D. melanogaster, C. elegans, P. falciparum, D. discoideum, A. thaliana, S. pombe, M. janaschii, D. rerio and H. sapiens analysed in this study were obtained from the AlphaFold2 database (https://alphafold.ebi.ac.uk; 24,25 ). To generate predicted models of human SNX25 including the previously annotated transmembrane domain 5,28 and the S. cerevisiae Lec1/Ypr097w to assess the flexibility of its PX domain, we used the AlphaFold2 neural-network 24 implemented within the freely accessible ColabFold pipeline 29 . For each modelling experiment ColabFold was executed using default settings where multiple sequence alignments were generated with MMseqs2 51 and structural relaxation of final peptide geometry was performed with Amber 52 to generate three models per structure.…”