Endogenous RNF185 was susceptible to proteinase K digestion, and similar result was obtained for Mfn1, while OPA1 and Tim23 remained resistant to proteinase K digestion because of the protection of mitochondrial membranes. To further characterize the topology of RNF185 on mitochondria, we made a construct with a Flag tag and a Myc tag expressed at the N-terminus and C-terminus of RNF185 protein respectively. Mitochondrial fractions isolated from those Flag- RNF185-Myc-expressing cells were subjected to proteinase K digestion at various time points. Both the Flag and Myc tags were readily susceptible to proteinase K digestion, and their signals weakened gradually and disappeared within 20 min of treatment.

A similar pattern was observed for the MOM protein Tom20. In contrast, cytochrome c and intermembrane space protein Tim23 remained intact. Taken together, our experimental evidence suggests a model for RNF185’s subcellular localization on mitochondria. Flag-RNF185-Myc is a MOM protein that crosses the membrane twice, exposing its RING domain and short C- terminus to the cytosol. HeLa cells with ectopic expression of RNF185 displayed abnormal morphology with globular, shrinking and punctate cell shape, which is usually found in dying cells[27]. However, the flow cytometric assay with Annexin V and 7-AAD did not show apoptosis in these cells. Moreover, over-expression of RNF185 caused cell cycle arrest and inhibited cell viability. A tight relationship between autophagy and cell cycle regulation is revealed by the recently emerging data[28,29]. To examine whether G1 arrest and abnormal cell shape induced by over-expression of RNF185 are related to autophagy, we performed LC3I to LC3II conversion assay, a typical and simple method to detect signs of autophagy[30]. As shown in Fig. 3B, increased levels of LC3II were observed in cells expressing RNF185 and cells treated with rapamycin or incubated inHank’s Buffered Salt Solution. Besides, knocking down RNF185 by siR-341 and siR-440 decreased the base level of LC3II. These consistent results suggest that RNF185 is associated with autophagy regulation. The development of autophagy is frequently assessed by the number and intensity of GFP-LC3 vesicles[31]. To verify whether LC3 is redistributed after over-expression of RNF185, we checked HeLa cells co-transfected with GFP tagged LC3 and RFP tagged RNF185. The characteristic redistribution of GFP-LC3 was observed, from a diffused cytoplasmic staining in control cells to punctate vesicular structures following over-expressing of RNF185. We also tested the function of RNF185’s two mutated forms for induction of autophagy by GFP-LC3 distribution assay.

The percentages of RPF positive cells with obviously punctate GFP-LC3 were greatly increased when wild type RNF185 was expressed, but not for empty vector, RING domain mutated RNF185 or TM domains deleted RNF185. These results suggested that the induction of punctate GFP-LC3 by over-expressed RNF185 is dependent on its intact RING domain and TM domains. To promote the degradation of their luminal content, autophagosomes fuse with lysosomes, thus forming the so-called autophagolysosomes[32]. We also detected the accumulation of possible autophagolysosomes using the lysosome membrane marker CD63. As shown in Fig. 4A, after overexpression of RNF185, GFP-LC3 accumulated dramatically and overlapped well with RFP tagged CD63. In addition, an increased accumulation of lysosome was observed in HeLa cells that were treated with rapamycin or transfected with expression construct for RNF185. A common intracellular stress that effectively leads to induction of autophagy is the formation of ROS. But our results indicated that HeLa cells over-expressed RNF185 had a relative lower level of ROS measured by DCFHDA staining. Mitochondria are the major sources generating ROS, which suggests that the reduced ROS may be caused by the loss of mitochondria mass. Indeed, we observed a dramatic loss of MitoTracker Red staining for the cells with very high level of RNF185, implying that the abundance of ectopic expressed RNF185 correlated with the degradation of mitochondria by autophagy.