Annexin A6 controls multi-organelle contact site formation and endolysosomal positioning, and remodels the STARD3 interactome

Annexin A6 (ANXA6) regulates cholesterol transfer across membrane contact sites (MCSs) between late endosomes/lysosomes (LE/Lys) and the endoplasmic reticulum (ER) via the late endosomal StAR-related lipid transfer domain-3 (STARD3) transporter. Here, we describe a significant reduction of MCSs in ANXA6-depleted HeLa cells, which could be rescued by restoration of ANXA6 expression. Using AnxA6 as bait in BioID-based assays, we demonstrate that ANXA6 interacts with various tethers and bona fide MCS proteins that can modulate multi-organelle contacts. STARD3 interactors identified in BioID assays include the mitochondrial translocator protein (TSPO) and myosin heavy chain 9 (MYH9). Strikingly, reduced MCS formation in ANXA6-depleted cells was associated with changes in the STARD3 interactome that indicate altered MCS tethering functions of STARD3. Specifically, ANXA6 deficiency correlated with (1) altered positioning of STARD3-positive LE/Lys; (2) a new repertoire of cortical actin-binding proteins, including myosins interacting with STARD3; (3) and decreased microvillar structures and focal adhesions.