Mavinakere Manohara S, Williamson Chad D, Goldmacher Victor S, Colberg-Poley Anamaris M
Center for Cancer and Immunology Research, Children's Research Institute, Room 5720, Children's National Medical Center, 111 Michigan Ave. NW, Washington, DC 20010, USA.
J Virol. 2006 Jul;80(14):6771-83. doi: 10.1128/JVI.00492-06.
The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.
人类巨细胞病毒（HCMV）UL37糖蛋白（gpUL37）会在内部发生切割，其切割产物分别转运至线粒体或保留在分泌途径中。为了确定gpUL37切割的必要条件，外显子3（UL37x3）内内质网（ER）信号肽酶I共有位点的-1和-3位残基被大体积酪氨酸取代（gpUL37切割位点突变体I）。该UL37x3突变体的内部切割受到抑制，证实了193/194位氨基酸处共有位点的使用。gpUL37切割位点突变体I的全长线粒体形式进行了N糖基化且对内切糖苷酶H敏感，表明在其线粒体导入之前发生了内质网转运和加工。此外，这些结果表明gpUL37的内部切割对其N糖基化并非必需。切割位点上游UL37疏水核心的部分缺失或破坏导致蛋白质丰度降低，表明UL37x3疏水α螺旋有助于gpUL37的正确折叠或稳定性。将UL37x3疏水核心和切割位点插入pUL37(M)（gpUL37的一个剪接变体，缺乏这些序列，既不进行蛋白水解切割也不进行N糖基化），导致其内部切割和N糖基化。其NH(2)末端片段pUL37(M-NH2)在线粒体中检测到的丰度更高，而其N糖基化的C末端片段gpUL37(M-COOH)主要在内质网中检测到，其方式类似于gpUL37切割产物。这些结果表明，UL37x3的178至205位氨基酸是gpUL37内部切割的先决条件，并改变了UL37蛋白拓扑结构，使其C末端序列能够进行N糖基化。相比之下，存在于pUL37x1、pUL37(M)和gpUL37中的NH(2)末端UL37x1疏水前导序列不会从成熟的UL37蛋白上切割下来，在运输过程中为UL37异构体保留一个膜锚定。综上所述，这些结果表明HCMV gpUL37经历了连续的运输过程，在此过程中它先进行内质网转运、加工，然后导入线粒体。
