In order to reveal the underlying molecular mechanism for RNF185 induced autophagy, we set out to identify its potential partners. Given theMOMlocalization of RNF185, we searched for Bcl-2 family members as they are mitochondria associated proteins and have emerged as regulators of autophagy. Since ATG5 is inducibly expressed at mitochondria during autophagy[37,38], we included ATG5 in our co-transfections consisting of 2HA tagged Bcl-2 family proteins with TM domains and 3Flag tagged RNF185. The results of co-immunoprecipitation clearly demonstrated that BNIP1 and ATG5 could be pulled down by RNF185. Likewise, RNF185 could also be pulled down by either BNIP1 or ATG5. The selfubiquitination of E3 ligase is demonstrable in vivo and can be used as a method to assay the ubiquitin E3 ligase activity of RING proteins[45,46,47]. First we found that 3Flag tagged RNF185 was intensively polyubiquitinated with endogenous ubiquitin or exogenous ubiquitin. The polyubiquitination of RNF185-RM was significantly decreased compared with wild type RNF185, suggesting that the E3 activity of RNF185 is RING domain dependent. Interestingly, the RNF185-TM mutant almost completely lost the activity of selfpolyubiquitination, implying that the mitochondrial localization is also critical for RNF185’s function as a ubiquitin E3 ligase. To assess whether RNF185 targets BNIP1 ubiquitination in vivo, Myc tagged ubiquitin was cotransfected with 2HA tagged RNF185 and 3Flag tagged BNIP1. Ectopically expressed RNF185 caused extensive polyubiquitination of BNIP1.
A low level of ubiquitination of BNIP1 was observed in the group without RNF185 transfection, presumably due to endogenous ubiquitin E3 ligases. Using ubiquitin mutants, we observed that BNIP1 was polyubiquitinated to a much lesser degree when the K63 of Mycubiquitin was mutated to R63. Therefore, BNIP1 was modified by K63-based polyubiquitin linkage, and this modification was consistent with the self-polyubiquitination pattern of RNF185.
The clearance of protein inclusions by autophagy was promoted by autophagy receptor p62, which preferentially partners with K63-linked polyubiquitin. The association of RNF185 with autophagy regulation and the polyubiquitination of BNIP1 through K63-linkage led us to assess the involvement of p62 in this pathway. Endogenous p62 was detected by western blot after the cotransfection of 3Flag tagged BNIP1, 2HA tagged RNF185 and Myc tagged ubiquitin or vector controls. As shown in Fig. 7E, p62 is co-immunoprecipitated with BNIP1. When both 2HA-RNF185 and Myc-Ub were over-expressed, BNIP1 could recruit much more p62, although endogenous RNF185 and endogenous ubiquitin also contributed to the interaction between p62 and polyubiquitinated BNIP1. In addition, we checked the endogenous localization of BNIP1 and p62 in HeLa cells. Alexa Fluor 488 conjugated endogenous BNIP1 and TRITIC conjugated endogenous p62 overlapped well in the cytoplasm, further providing the locational evidence for the recruitment of p62 by BNIP1. Mitochondria are essential for a variety of cellular functions, including ATP production, lipid biosynthesis, and calcium homeostasis. Recent investigations indicate that certain aspects of mitochondrial functions, including mitochondrial protein quality control and membrane dynamics, are regulated by the ubiquitinconjugation system[52]. Both MARCH5(RNF153)[53,54] and MULAN(RNF218)[5], two MOM ubiquitin E3 ligases clearly described so far, were found to be involved in the regulation of mitochondria dynamics. Unlike these MOM E3 ligase, RNF185 does not affect mitochondria fusion and fission; whereas RNF185 functions as a specific regulator for autophagy of the mitochondria. The mechanism for the mitochondrial homeostasis by autophagy remained largely unknown.