An Inner Mitochondrial Membrane Microprotein from the SLC35A4 Upstream ORF Regulates Cellular Metabolism

[Display omitted] •Upstream Open Reading Frames (uORFs) regulate translation, but can they encode functional proteins?•We characterized a microprotein encoded by the uORF of SLC35A4 mRNA.•We found that this microprotein contains a single-pass transmembrane that localizes it to the inner mitochondrial membrane.•Loss of function studies showed that the SLC35A4 microprotein (SLC35A4-MP) regulates cellular respiration.•There are hundreds of uORFs that encode conserved microproteins that are likely to have independent functions in biology. Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing downstream translation under stress conditions. Computational and empirical methods have identified uORFs in the 5′-UTRs of approximately half of all mouse and human transcripts, making uORFs one of the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs were rarely studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functional microprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add SLC35A4-MP to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.