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J. Biol. Chem., Vol. 283, Issue 49, 33826-33837, December 5, 2008

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The Cotranslational Maturation Program for the Type II Membrane Glycoprotein Influenza Neuraminidase^*

Ning Wang¹, Emily J. Glidden¹, Stephanie R. Murphy, Bradley R. Pearse², and Daniel N. Hebert³

From the Department of Biochemistry and Molecular Biology and the Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N- and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regio-specific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

Received for publication, September 5, 2008 , and in revised form, October 10, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Chemistry-Biology Interface Training Grant T32GM00815 (to B. R. P.). This work was also supported by U. S. Public Health Service Grant CA79864 (to D. N. H.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text and a supplemental figure.

¹ Both authors contributed equally to this work.

² Supported in part by a predoctoral University fellowship from the University of Massachusetts.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Massachusetts, 710 N. Pleasant St., Amherst, MA 01003. Tel.: 413-545-0079; Fax: 413-545-3291; E-mail: dhebert{at}biochem.umass.edu.

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