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J. Biol. Chem., Vol. 280, Issue 1, 261-269, January 7, 2005

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Differential Stability of Biogenesis Intermediates Reveals a Common Pathway for Aquaporin-1 Topological Maturation^*

Teresa M. Buck and William R. Skach

From the Molecular Medicine Division, Department of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

Topological studies of multi-spanning membrane proteins commonly use sequentially truncated proteins fused to a C-terminal translocation reporter to deduce transmembrane (TM) segment orientation and key biogenesis events. Because these truncated proteins represent an incomplete stage of synthesis, they transiently populate intermediate folding states that may or may not reflect topology of the mature protein. For example, in Xenopus oocytes, the aquaporin-1 (AQP1) water channel is cotranslationally directed into a four membrane-spanning intermediate, which matures into the six membrane-spanning topology at a late stage of synthesis (Skach, W. R., Shi, L. B., Calayag, M. C., Frigeri, A., Lingappa, V. R., and Verkman, A. S. (1994) J. Cell Biol. 125, 803–815 and Lu, Y., Turnbull, I. R., Bragin, A., Carveth, K., Verkman, A. S., and Skach, W. R. (2000) Mol. Biol. Cell 11, 2973–2985). The hallmark of this process is that TM3 initially acquires an Nexo/Ccyto (Type I) topology and must rotate 180° to acquire its mature orientation. In contrast, recent studies in HEK-293 cells have suggested that TM3 acquires its mature topology cotranslationally without the need for reorientation (Dohke, Y., and Turner, R. J. (2002) J. Biol. Chem. 277, 15215–15219). Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.

Received for publication, August 30, 2004 , and in revised form, October 27, 2004.

^* This work was supported by National Institutes of Health Grants GM53457 and DK51818, an American Heart Association Established Investigator Grant (to W. R. S.), and National Institutes of Health Grant HL007781 (to T. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Molecular Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-7322; Fax: 503-494-7368; E-mail: skachw{at}ohsu.edu.

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