Shuttling of Membrane-Bound Cytochrome B5 between Mitochondria and ER

Abstract

Cytochrome b5 type B has long been described as a tail-anchored protein of the outer mitochondrial membrane (cb5-mito). Using a fusion protein consisting of photoactivatable GFP fused to the C-terminus membrane-targeting segment of cb5-mito, we have found that while this construct concentrates in the mitochondrial membrane, it is continuously exchanged between the membranes of the mitochondrion and the endoplasmic reticulum. This effect does not depend on the presence of mitofusin 1 or 2, as it occurs in Mfn-null MEFs as well as in wild type cells. Interestingly, a point mutation in the hydrophobic, membrane-targeting sequence of cb5-mito that was recently reported to prevent its association with autophagosome membranes, also eliminates its exchange with the ER. While lipids and viral proteins have been shown to traffic from ER to mitochondria, this is, to our knowledge, the first demonstration of such a back-and-forth shuttling, representing both a new aspect of the ER-mitochondrial interface and an Mfn-independent pathway for the exchange of an outer mitochondrial membrane protein.