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J. Biol. Chem., Vol. 280, Issue 16, 16402-16409, April 22, 2005

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Endoplasmic Reticulum Localization of Gaa1 and PIG-T, Subunits of the Glycosylphosphatidylinositol Transamidase Complex^*

Saulius Vainauskas and Anant K. Menon

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544

After integration into the endoplasmic reticulum (ER) membrane, ER-resident membrane proteins must be segregated from proteins that are exported to post-ER compartments. Here we analyze how human Gaa1 and PIG-T, two of the five subunits of the ER-localized glycosylphosphatidylinositol transamidase complex, are retained in the ER. Neither protein contains a known ER localization signal. Gaa1 is a polytopic membrane glycoprotein with a cytoplasmic N terminus and a large luminal loop between its first two transmembrane spans; PIG-T is a type I membrane glycoprotein. To simplify our analyses, we studied Gaa1 and PIG-T constructs that could not interact with other subunits of the transamidase. We now show that Gaa1₂₈₂, a truncated protein consisting of the first TM domain and luminal loop of Gaa1, is correctly oriented, N-glycosylated, and ER-localized. Removal of a potential ER localization signal in the form of a triple arginine cluster near the N terminus of Gaa1 or Gaa1₂₈₂ had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1₂₈₂ appended to 2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1₂₈₂, and by implication Gaa1, does not contain any dominant ER-sorting determinants. The data suggest that Gaa1 is passively retained in the ER by a signalless mechanism. In contrast, similar analyses of PIG-T revealed that it is ER-localized because of information in its transmembrane span; fusion of the PIG-T transmembrane span to Tac antigen, a plasma membrane-localized protein, caused the fusion protein to remain in the ER. These data are discussed in the context of models that have been proposed to account for retention of ER membrane proteins.

Received for publication, December 20, 2004 , and in revised form, February 7, 2005.

^* This work was supported by National Institutes of Health Grant GM55427. 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: Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-263-2636 (ext. 3468); Fax: 608-262-3453; E-mail: saulius{at}biochem.wisc.edu.

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