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

Saulius Vainauskas,Anant K Menon

doi:10.1074/jbc.m414253200

Abstract

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 Gaa1(282), 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 Gaa1(282) had no effect on ER localization. Fusion proteins consisting of different elements of Gaa1(282) appended to alpha2,6-sialyltransferase or transferrin receptor could exit the ER, indicating that Gaa1(282), 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.

Highlights

The mechanism(s) by which ER1-resident proteins are sorted from those destined for post-endoplasmic reticulum (ER) compartments is a subject of ongoing debate [1,2,3,4,5]
Recent data suggest that Gaa1 and possibly PIG-U may play a role in recruiting GPI to GPI transamidase (GPIT) [28, 35], and it has been proposed that a ␤-propeller structure in the luminal domain of PIG-T gates access to the active site of Gpi8 [36]
We made a T545 construct (Fig. 5A, middle; compare with the image for intact, epitope-tagged PIG-T in the top panel) in which the C-terminal 33 amino acids were replaced with a V5 epitope tag. (We previously showed that the membrane-proximal cytoplasmic display of the V5 tag does not cause ER localization of proteins destined for post-ER compartments [38]; see below.) When transiently overexpressed in HeLa cells, the recombinant protein was found located only in the ER, suggesting that the C-terminal sequence is not required for ER localization (Fig. 5A, middle)

Summary

Introduction

The mechanism(s) by which ER1-resident proteins are sorted from those destined for post-ER compartments is a subject of ongoing debate [1,2,3,4,5]. Some ER residents, like ER chaperones, may be continuously engaged with protein traffic entering the ER such that their own exit is impeded These interactions or the numerous quasi-interactions that are probably promoted by the high protein density in the ER may be sufficient to retain proteins if the rate of bulk flow of membrane and fluid departing the ER is low and if the proteins lack explicit export signals. We classify these possibilities under the heading of “signalless” retention mechanisms. Recent studies show that human Gaa has a cytoplasmically oriented N terminus and spans the ER membrane seven times [37]; the membrane topology of PIG-S and PIG-U remains to be experimentally determined

Methods

Results

Conclusion