Identifying targeting signals and distinct localization-based roles of yeast aldehyde dehydrogenase Hfd1

Hfd1 is a yeast fatty aldehyde dehydrogenase that catalyzes the oxidation of long-chain aliphatic aldehydes and localizes to membranes in multiple organelles, including mitochondria, the endoplasmic reticulum (ER), and lipid droplets. Here, we identified the segments of Hfd1 responsible for this multiple-organelle targeting and generated variants that localize exclusively to mitochondria, the ER, or the cytosol. These Hfd1 variants allowed us to reveal the relationship between the subcellular localization and the function of Hfd1 in coenzyme Q biosynthesis and sphingolipid metabolism. The catalytic N-terminal domain of Hfd1, when exposed to the cytosol, is sufficient to support coenzyme Q biosynthesis regardless of whether Hfd1 has an anchor or is anchored to either the mitochondrial or ER membranes. Furthermore, Hfd1 contributes to the mitigation of reactive oxygen species and to the oxidation of hexadecanal and hexadecenal, which may be important for efficient mitochondrial protein transport and/or quality control in cooperation with Ubx2 and the TOM complex.