To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

The membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Immunofluorescence staining of COS-1 cells transfected with cDNA encoding ANKRD22 coupled with organelle markers revealed that ANKRD22 localized specifically to lipid droplets (LD). Analysis of the intracellular localization of ANKRD22 mutants C-terminally fused to glycosylatable tumor necrosis factor (GLCTNF) and assessment of their susceptibility to protein N-glycosylation revealed that ANKRD22 is synthesized on the endoplasmic reticulum (ER) membrane as an N-myristoylated hairpin-like monotopic membrane protein with the amino- and carboxyl termini facing the cytoplasm and then sorted to LD. Pro98 located at the center of the predicted membrane domain was found to be essential for the formation of the hairpin-like monotopic topology of ANKRD22. Moreover, the hairpin-like monotopic topology, and positively charged residues located near the C-terminus were demonstrated to be required for the sorting of ANKRD22 from ER to LD. Protein N-myristoylation was found to positively affect the LD localization. Thus, multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD.