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J. Biol. Chem., Vol. 277, Issue 17, 15215-15219, April 26, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/17/15215    most recent C100646200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dohke, Y. Articles by Turner, R. J. Search for Related Content PubMed PubMed Citation Articles by Dohke, Y. Articles by Turner, R. J. Social Bookmarking                      What's this?

Evidence That the Transmembrane Biogenesis of Aquaporin 1 Is Cotranslational in Intact Mammalian Cells^*

Yoko Dohke and R. James Turner

From the Membrane Biology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Most polytopic membrane proteins are believed to integrate into the membrane of the endoplasmic reticulum (ER) cotranslationally. However, recent studies with Xenopus oocytes and dog pancreatic microsomes have suggested that this is not the case for human aquaporin 1 (AQP1). These experiments indicate that membrane-spanning segments (MSSs) 2 and 4 of AQP1 do not integrate into the membrane cotranslationally so that this protein initially adopts a four MSS topology. A later maturation event involving a 180-degree rotation of MSS 3 from an N_lum/C_cyt to an N_cyt/C_lum orientation and the concomitant integration of MSSs 2 and 4 into the membrane results in the final six MSS topology. Here we examine the biogenesis of AQP1 in the human embryonic kidney cell line HEK-293T. To do this, we constructed an expression vector for a fusion protein consisting of the enhanced green fluorescent protein followed by an insertion site for AQP1 sequences and a C-terminal glycosylation tag. We then transiently transfected HEK-293T cells with this vector containing the AQP1 sequence truncated after each MSS. Glycosylation of the C-terminal tag was used to monitor its location relative to the ER lumen and consequently the membrane integration and orientation of successive MSSs. In contrast to previous studies our results indicate that AQP1 integrates into the ER membrane cotranslationally in intact HEK-293T cells.

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