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Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells

Petros Bozidis,^1, Chad D. Williamson,^1,2 and Anamaris M. Colberg-Poley^1,2,3*

Center for Cancer and Immunology Research, Children's Research Institute, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010,¹ Department of Biochemistry and Molecular Biology, George Washington University, Washington, DC 20037,² Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC 20037³

Received 14 November 2007/ Accepted 21 December 2007

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 antiapoptotic 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 the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). 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 subcellular 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.

Published ahead of print on 16 January 2008.

Present address: Laboratory of Biology, Medical School, University of Ioannina, 45500 Ioannina, Greece.