Abstract
Previously, we reported that Centrosomal P4.1-associated protein (CPAP/CENPJ/SAS-4) positively regulates multivesicular body (MVB) biogenesis and endocytic vesicular transport (EVT). Here, we show that CPAP is required for Rab5-to-Rab7 conversion, and recruitment of TSG101 to early endosome (EE) is the molecular mechanism by which CPAP promotes MVB formation. CPAP depletion disrupts Rab7 and TSG101 recruitment to EE and blocks endosome maturation. While endogenous CPAP co-precipitates with ESCRT-proteins TSG101, HRS and ALIX, exogenously expressed CPAP only interacts with TSG101. CPAP localizes to endosome and colocalizes with HRS and TSG101 during EVT progression. TSG101 recruitment to endosome, Rab5-to-Rab7 conversion, and EVT of EGFR to MVB in CPAP-depleted cells are restored by re-introduction of CPAP or overexpression of, but not endogenous, HRS. These observations show that CPAP is an ESCRT-associated protein, which functions upstream of TSG101, but in parallel to HRS, during MVB formation and EVT of cargo to the lysosome. HIGHLIGHTS: CPAP-deficiency prevents Rab5-Rab7 conversion and TSG101 recruitment to early endosomes during MVB formationCPAP is an essential ESCRT proteinCPAP functions parallel to HRS during MVB formationCPAP-TSG101 interaction is essential for Rab5-Rab7 conversion and endosome maturation.