The Bro1-related protein HD-PTP/PTPN23 is required for endosomal cargo sorting and multivesicular body morphogenesis

TLDR

In yeast, Bro1p is essential for directing endocytic cargo into multivesicular bodies, but the mammalian counterpart is unknown and Alix, though structurally similar, has not been shown to participate in MVB formation. The study aimed to determine whether Alix or related proteins are involved in endosomal cargo sorting. To test this, the authors engineered a reporter receptor fused to a retroviral peptide that binds Alix, enabling assessment of its sorting behavior. The Alix‑binding chimera was efficiently sorted to late endosomes, and its trafficking required the Alix‑related protein HD‑PTP; depletion of HD‑PTP impaired lysosomal delivery of fluid‑phase markers and EGF receptor, accumulated ubiquitinated proteins on endosomes, disrupted MVB morphology, and rescue with an RNAi‑resistant HD‑PTP or mutants confirmed that the Bro1 domain and ESCRT‑III binding are essential for function.

Abstract

The Saccharomyces cerevisiae protein Bro1p is required for sorting endocytic cargo to the lumen of multivesicular bodies (MVBs). The mammalian ortholog of Bro1p is not known; although Alix, a structurally related protein, supports the topologically similar process of virus budding, functional studies have so far failed to identify a role for Alix in MVB formation. To establish whether Alix or similar protein(s) participate in endosomal sorting, we attached a retroviral peptide that binds Alix to a reporter receptor. This chimera was sorted efficiently away from the early endosome to the lumen of late endocytic compartments. Surprisingly, sorting was not prevented by depleting Alix but instead required the Alix-related protein His domain phosphotyrosine phosphatase (HD-PTP)/His-Domain/Type N23 protein tyrosine phosphatase (PTPN23). Depletion of HD-PTP also reduced transfer of fluid-phase markers and EGF receptor to lysosomes, caused the accumulation of ubiquitinated proteins on endosomal compartments and disrupted the morphogenesis of MVBs. Rescue experiments using an RNAi-resistant version of HD-PTP and HD-PTP mutants demonstrated an essential role for the HD-PTP Bro1 domain, with ESCRT-III binding correlating with full biological activity.