Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondria associated membranes of human cells

Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010; Department of Biochemistry and Molecular Biology, George Washington University, Washington DC 20037; and Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington DC 20037

^* To whom correspondence should be addressed. Email: acolberg-poley{at}cnmcresearch.org.

	Abstract

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent anti-apoptotic protein, which prevents cytochrome C release from mitochondria. The UL37x1 NH₂-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from ER to mitochondria through direct contacts between the two organelles, provided by mitochondria-associated membranes (MAM). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase-1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using sub-cellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37_NH2 and gpUL37_COOH were both detected in the ER and MAM fraction, even though only pUL37_NH2 is preferentially imported into mitochondria but gpUL37_COOH is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH₂-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.