Tom40 import and TOM complex assembly kinetics in subsarcolemmal and intermyofibrillar mitochondria

Abstract

The insertion of nuclear-derived proteins into mitochondria is facilitated by the translocases of the outer membrane (TOM) complex. Tom40 is the main component of this complex. Its import and assembly has not been characterized in skeletal muscle. Thus, our purpose was to compare the import and assembly of Tom40 in muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Tom40 protein levels were not different between SS and IMF mitochondria. However, Tom40 import into the outer membrane was higher (p<0.05) in SS mitochondria at all time points (5, 10, 20 min) when compared to IMF mitochondria. Tom40 assembly was monitored using blue native electrophoresis. Tom40 first assembles into a ~250kDa complex (Intermediate I), followed by a ~120 kDa complex (Intermediate II), before its final insertion into the ~380 kDa TOM complex. In IMF mitochondria, 70–80% of Tom40 was incorporated into Intermediate I by 5 min, whereas only 40–50% was found in Intermediate I in SS mitochondria. However, the sequential incorporation of Tom40 into Intermediate II was less rapid in IMF mitochondria, and only 45% of Tom40 was incorporated into the final TOM complex by 60 min. In contrast, SS mitochondria incorporated 65–70% of Tom40 by 60 min. Thus, despite similar levels of steady state Tom40 protein content, the import and assembly of Tom40 into the TOM complex differs among muscle mitochondrial subtypes.