MNP33 is a novel mitochondrial protein that reprograms cellular bioenergetics and lipid metabolism

Genomic regions previously annotated as non-coding are now recognized sources of functional microproteins, termed the "dark proteome", though their identification and characterization remain challenging. Addressing this, we developed a novel proteomic strategy combining lipid droplet (LD) enrichment with mass spectrometry against the database of sORF-derived proteins. This approach yielded the first reported LD-resident microprotein, LDANP1, and subsequently revealed other candidates from the same screen, such as MNP33, were localized to mitochondria. Here, we provide the first in-depth functional characterization of MNP33, a novel 28-amino acid protein encoded by an sORF within the Cdhr4 gene locus. We confirm MNP33 localizes to the inner mitochondrial membrane and demonstrate its interaction with the adenine nucleotide translocase 2 (ANT2). Functionally, MNP33 expression remodels mitochondrial bioenergetics, increasing basal respiration and proton leak while paradoxically elevating membrane potential, partly through modulating ANT2 activity linked to enhanced glycolysis. Furthermore, MNP33 shifts cellular lipid metabolism, favoring cholesteryl ester storage over triacylglycerols via stabilization of ACAT1, and is associated with closer ER-mitochondria contacts. MNP33 also induces ROS production and autophagy while inhibiting cell proliferation without increasing apoptosis. This study establishes MNP33 as a novel mitochondrial regulator emerging from the dark proteome, providing a mechanistic link between non-coding genetic elements, ANT2 function, mitochondrial bioenergetics, cellular metabolism, and stress responses. Our work highlights functional importances of the dark proteins and validates organelle-focused proteomics for discovering key cellular regulators.