ESCRT-I controls lysosomal membrane protein homeostasis and restricts MCOLN1-dependent TFEB/TFE3 signaling.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of membrane proteins from limiting membranes of late endosomes. Recent studies revealed that yeast ESCRT proteins also sort for degradation, ubiquitinated proteins from vacuolar membrane. However, whether mammalian ESCRTs perform similar function at lysosomes remained unknown. Here, we studied the involvement of mammalian ESCRT-I in maintaining lysosomal membrane homeostasis and its implication in lysosome-related signaling. We show that ESCRT-I restricts the size of lysosomes and promotes degradation of lysosomal Ca2+ channel, MCOLN1 protein. ESCRT-I depletion induced transcriptional response related to MiT-TFE signaling and cholesterol biosynthesis, pointing to lysosomal dysfunction. The lack of ESCRT-I promoted abnormal cholesterol accumulation on lysosomes and activated TFEB/TFE3 transcription factors in Ca2+-MCOLN1-dependent, but lipid-independent manner. Hence, our study provides evidence that ESCRT-I maintains lysosomal homeostasis and elucidates MiT-TFE regulatory mechanism activated in response to ESCRT-I deprivation The aim of the study was to analyze by RNA sequencing the transcriptional changes upon single siRNA-mediated depletion of two core component of ESCRT-I complex, Tsg101 or Vps28 subunit. As a model, human colorectal RKO cell line was used. 3 biological replicates were prepared for each experimental condition. The conditions were: 1/RKO transfected with non -targeting siRNA (siCtrl#1), 2/ RKO transfected with non-targeting siRNA (siCtrl#2), 3/ RKO transfected with siRNA targeting TSG101, sequence #1 (siTsg101#1), 4/ RKO transfected with siRNA targeting TSG101, sequence #2 (siTsg101#2), 5/ RKO transfected with siRNA targeting VPS28, sequence #1 (siVps28#1), 6/ RKO transfected with siRNA targeting VPS28, sequence #2 (siVps28#2). siCtrl#1-transfected cells served as the reference sample.