Functional analysis of Miro GTPase domains in the mitochondrial motor adaptor complex

Abstract

Mitochondria are actively transported in all somatic cells where their motility is essential for proper cellular function. Mitochondrial transport relies on microtubules and a highly regulated motor/adaptor complex that consists of Miro1, a mitochondrial outer membrane protein; TRAK1/2, the adaptor proteins; and kinesin-1, and dynein, the molecular motors. Miro1 and its family member Miro2 are Rho like GTPases that contains two GTPase domains (N-GTPase and C- GTPase) that are separated by EF hands. Although the GTPase domains of Miro are a prominent feature, their functional significance is controversial. During the course of my thesis, I have uncovered a novel mechanism by which the Miro1 N-GTPase regulates assembly of the motor/adaptor complex. Although Miro2 has long been assumed to play a similar role to Miro1 in mitochondrial motility, I have found that Miro2 does not interact with components of the motor/adaptor complex in the same way as Miro1. To study the function of the Miro GTPase domains without confounding influences from the presence of endogenous mitochondrial Miro, I’ve misdirected Miro1 and Miro2 to peroxisomes with the transmembrane domain of PEX3. When PEX3-Miro1 is co-expressed with components of the motor/adaptor complex, TRAK1/2 and the motors kinesin-1 and dynein (P135), these proteins are also re-directed to peroxisomes. Using the peroxisome-directed Miro1, we find that the Miro1 N- GTPase regulates the ability of Miro1 to interact with the rest of the motor adaptor complex: neither TRAK1/2 nor kinesin or P135 colocalize on peroxisomes with the GDP-locked (T18N) mutation. In addition to a loss of interaction with the motor/adaptor complex, the distribution of peroxisomes in COS-7 cells and hippocampal neurons are correspondingly affected. The complex assembles correctly with the GTP-locked N-GTPase (P13V) mutation. In contrast, neither the GTP-locked nor GDP-locked C-GTPase mutations substantially alter Miro1 recruitment of TRAK1/2 or kinesin to peroxisomes. We find that PEX3-Miro2 is unable to recruit the components of the motor/adaptor complex to peroxisomes regardless of the state of its N-terminal or C-terminal GTPase. Thus, N-GTPase of Miro1 is critical for regulating Miro1’s interaction with the other components of the motor/adaptor complex and thereby for regulating mitochondrial motility.