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Understanding ER-to-Mitochondria trafficking of proteins using human cytomegalovirus UL37 proteins Open Access

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While precise protein sorting to cellular compartments can be directed through the ER and trans-Golgi networks, mitochondrial proteins curiously avoid this secretory pathway machinery entirely. Typically, nuclear-encoded mitochondrial proteins derive from mRNA translated on cytosolic ribosomes, which then post-translationally incorporate into the mitochondria, directed by mitochondria-specific amino acid sequences. Paradoxically pUL37x1, a type I transmembrane viral protein essential for human cytomegalovirus growth in vitro and in vivo, contains a signal sequence enabling it to divergently traffic to the ER as well as to the mitochondria. This bipartite sequence is comprised of an NH2-terminal stretch of hydrophobic residues and a juxtaposed downstream domain. The hydrophobic region consisting of amino acids 2-23 is important for incorporation of pUL37x1 into both ER and mitochondrial membranes, while the downstream amino acids 23-34 are uniquely required for efficient mitochondrial localization. The ability of pUL37x1 to localize to the mitochondria, and there inhibit apoptosis, assuredly confers a favorable advantage to the virus. Hence the anti-apoptotic capability of pUL37x1 is a copiously studied topic of the HCMV field. Intriguingly, however, both laboratory and clinical HCMV strains display stringent conservation of the bipartite leader sequence of pUL37x1. This subtly, but powerfully, suggests the protein's ability to bifurcate its subcellular localization to both mitochondrial and secretory organelles has important functional consequences for viral infection.These studies aimed to define the relationship between ER- and mitochondria-localized UL37 protein species, as well as identify the amino acid determinants, within the leader sequence, responsible for the unique trafficking abilities of UL37 proteins. To accomplish this, site-directed genetic mutations were generated within UL37 protein isoforms and analyzed for subcellular localization using a range of fractionation techniques, as well as confocal microscopy. Data presented in this thesis reveal a sequential trafficking pathway utilized by UL37 proteins from ER sites of synthesis to mitochondrial outer membranes, and suggest a complex and reiterative mechanism of directing UL37 proteins to this pathway. Accumulation of UL37 proteins within mitochondria-associated membranes, a site of lipid transfer between ER and mitochondria, was detected. These ER subdomains were demonstrated to be credible sites of protein transfer between ER and mitochondrial membranes. Furthermore, a cholesterol binding domain was identified within the UL37 leader sequence, and subsequently linked to the intracellular lipid raft association of UL37 proteins. Lastly, kinetic data exposed a dramatically rapid importation of UL37 proteins into mitochondria.

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