Pex19 and cytosolic Hsp70 are involved in the import of mitochondrial tail- anchored proteins

Abstract

The Majority of mitochondrial proteins are synthetized in the cytosol and afterwards targeted to the organelle. The transport process is accompanied by cytosolic factors, which ensure targeting specificity and prevent the proteins from aggregation in the aqueous environment of the cytosol. This especially applies to tail-anchored (TA) proteins that are directed to membranes in a post-translational manner. Tail-anchored proteins are embedded into their corresponding membrane via a single transmembrane segment at their C-terminus whereas the majority of the protein is facing the cytosol. The targeting pathways of these proteins to the ER or to peroxisomes have been characterized. However, so far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane have not been found. Equally elusive remains the existence of mitochondrial membrane insertases or receptors for TA import. Thus, it is currently postulated that import of mitochondrial TA proteins is mediated solely by unassisted insertion without the requirement of any protein factors. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1, its co-chaperone Sti1, and the peroxisome import factor Pex19 as mediators of import of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (i) growth defect under respiration conditions; (ii) alteration in mitochondrial morphology; (iii) reduced steady-state levels of the mitochondrial single span proteins Fis1, Gem1, and Atg32; and (iv) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, we demonstrate that recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1 and that all three proteins share a mitochondrial and peroxisomal dual localization. Alteration in Atg32 levels are dependent on the mitochondrial receptors Mim1 and Tom20 suggesting that both can mediate Pex19 binding to mitochondria. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19.