Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.

The tumor natural killer (NK) cell line YTS was used to examine the cytoskeletal rearrangements required for cytolysis. A multiprotein complex weighing ∼1.3 mD and consisting of WASp-interacting protein (WIP), Wiskott-Aldrich syndrome protein (WASp), actin, and myosin IIA that formed during NK cell activation was identified. After induction of an inhibitory signal, the recruitment of actin and myosin IIA to a constitutive WIP–WASp complex was greatly decreased. Both actin and myosin IIA were recruited to WIP in the absence of WASp. This recruitment correlated with increased WIP phosphorylation, which was mediated by PKCθ. Furthermore, the disruption of WIP expression by WIP RNA interference prevented the formation of this protein complex and led to almost complete inhibition of cytotoxic activity. Thus, the multiprotein complex is important for NK cell function, killer cell immunoglobulin-like receptor inhibitory signaling affects proteins involved in cytoskeletal rearrangements, and WIP plays a central role in the formation of the complex and in the regulation of NK cell activity.