Cytosolic factor- and TOM-independent import of C-tail-anchored mitochondrial outer membrane proteins

Abstract

C‐tail‐anchored (C‐TA) proteins are anchored to specific organelle membranes by a single transmembrane segment (TMS) at the C‐terminus, extruding the N‐terminal functional domains into the cytoplasm in which the TMS and following basic segment function as the membrane‐targeting signals. Here, we analyzed the import route of mitochondrial outer membrane (MOM) C‐TA proteins, Bak, Bcl‐XL, and Omp25, using digitonin‐permeabilized HeLa cells, which provide specific and efficient import under competitive conditions. These experiments revealed that (i) C‐TA proteins were imported to the MOM through a common pathway independent of the components of the preprotein translocase of the outer membrane, (ii) the C‐TA protein‐targeting signal functioned autonomously in the absence of cytoplasmic factors that specifically recognize the targeting signals and deliver the preproteins to the MOM, (iii) the function of a cytoplasmic chaperone was required if the cytoplasmic domains of the C‐TA proteins assumed an import‐incompetent conformation, and intriguingly, (iv) the MOM‐targeting signal of Bak, in the context of the Bak molecule, required activation by the interaction of its cytoplasmic domain with VDAC2 before MOM targeting.