TRAK proteins encode distinct MIRO-dependent and MIRO-independent mechanisms for associating with the mitochondrial outer membrane

Abstract

I.AbstractMitochondria are essential to proper cell function, mislocalization of mitochondria leads to disease. Previous research has indicated that MYO19, an unconventional myosin, localizes to the mitochondria outer membrane (MOM) and enables actin-based mitochondrial movement along the cytoskeleton. MOM insertion of MYO19 is assisted by the small GTPase MIRO, a “molecular switch” that facilitates MYO19/mitochondria interactions. MIRO serves as a “recruiter” of MYO19 to the MOM, rather than the tether/receptor that mediates attachment, as MYO19 contains a second, MIRO-independent mitochondrial association domain. MIRO proteins have previously been reported to serve as attachment points for the microtubule-based motors through interactions with the adaptor protein, TRAK. Past research has identified a MIRO-binding domain of TRAK (TRAKMBD) that directly participates in the interaction with MIRO. We chose to investigate whether the interactions between TRAK and MOM paralleled our hypothesized mechanism for MYO19/MOM interactions by examining the MIRO-mediated enhancement of TRAK protein localization to mitochondria, by identifying the location of a MIRO-independent mitochondrial association domain in the c-terminus of TRAK proteins, and by examining the steady-state binding kinetics of various TRAK constructs to mitochondria. We interpret these data to indicate that MIRO proteins serve in the initial recruitment of TRAK proteins to mitochondria, but that the MIRO-independent domain plays a significant role in long-term association between TRAK and MOM.