MSTO1 Links Cytosolic TRiC Chaperone Activity to Mitochondrial Function

Abstract

Mitochondria are constantly fusing, dividing, and moving on microtubules to maintain structural and functional integrity. Mitochondrial dynamics are regulated by cytosolic factors and signaling pathways, providing critical integration into cellular physiology. In addition, maintenance of mitochondrial DNA (mtDNA) is essential for mitochondrial health, as the genome encodes essential oxidative phosphorylation proteins. Recently, mutations in MSTO1 have been linked to clinical disease phenotypes typical of mitochondrial dysfunction. MSTO1 is tubulin/FtsZ related protein that localizes to the cytoplasm. MSTO1 mutant patient-derived fibroblasts have short mitochondria and a striking loss of mtDNA. This suggests MSTO1 is a novel regulator of mitochondrial fusion and mtDNA maintenance. Outside of MSTO1 patient studies, virtually nothing is known about the function of MSTO1 in vertebrates. In Drosophila melanogaster, the MSTO1 homolog Misato has been shown to stabilize the TCP-1 (TRiC in humans) chaperone. TRiC is a heterooligomer that aids in the folding of about 10% of cytosolic proteins, with an obligate role in folding tubulin and actin. It is not known if this relationship between TRiC and MSTO1 is conserved in humans. To build a system to uncover the molecular details of MSTO1 in humans, I generated cell lines using the auxin-inducible degradation (AID) system to allow temporal control of MSTO1 protein stability. After two hours of auxin treatment, MSTO1-FLAG-AID protein decreased to almost undetectable levels. MSTO1-FLAG-AID cells treated with auxin for six days possessed shorter mitochondria than controls and MSTO1-FLAG-AID cells treated with auxin for only three days. MSTO1-FLAG-AID also did not interact with either Mfn1 or Mfn2 by co-immunoprecipitation. The extended length of time required for structural changes in the mitochondrial network and absence of mitofusin interaction suggest MSTO1 may indirectly regulate mitochondrial fusion. Furthermore, MSTO1- depleted cells had a significant decrease in TRiC protein expression 2 days before mitochondrial changes were observed. Knockdown of TRiC was sufficient to induce cells with a short mitochondrial network, suggesting the mitochondrial changes in MSTO1-depleted cells may be due to a loss of TRiC rather than MSTO1. Knockdown of TRiC also decreased MSTO1 protein levels, indicating that MSTO1 and TRiC protein levels are dependent on each other. Using co-immunoprecipitation and BN-PAGE, I found that MSTO1 interacts with the fully assembled TRiC. Using co-immunoprecipitation and BN-PAGE, we found that MSTO1 interacts with the fully assembled TRiC chaperone and our data suggests that MSTO1 assists in assembling early TRiC assembly intermediates. Together, these findings identify MSTO1 as a TRiC assembly factor and connect mitochondrial defects caused by MSTO1-depletion to the loss of TRiC.