25 26Recent studies in eukaryotes have demonstrated the translation of alternative open 27 reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We 28show that a large number of small proteins could in fact be coded by altORFs. The 29 putative alternative proteins translated from altORFs have orthologs in many species and 30 evolutionary patterns indicate that altORFs are particularly constrained in CDSs that 31 evolve slowly. Thousands of predicted alternative proteins are detected in proteomic 32 datasets by reanalysis using a database containing predicted alternative proteins. Protein 33 domains and co-conservation analyses suggest a potential functional relationship between 34 small and large proteins encoded in the same genes. This is illustrated with specific 35 examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein 36 encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting 37 mitochondrial fission. Our results suggest that many coding genes code for more than one 38 protein that are often functionally related.