Abstract
The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.
Highlights
Protein maturation and quality control in the endoplasmic reticulum (ER) involves a network of molecular chaperones, foldases, and sorting factors that are recruited at specific stages to optimize the folding process and monitor the integrity of the protein product (Ellgaard et al, 1999; Hebert and Molinari, 2007)
Transient nonnative conformations of HA trigger transitory GT1 reglucosylation, whereas terminal nonnative mutations induced long-term recognition. These results demonstrate that in the intact mammalian ER, GT1 acts posttranslationally to direct differential lectin chaperone binding through detection of slow folding and malformed regions of substrate glycoproteins
To separate monoglucosylated glycans produced via GT1 activity from those generated by glucosidase activity, the CHO mutant cell line MI8-5 was used
Summary
Protein maturation and quality control in the ER involves a network of molecular chaperones, foldases, and sorting factors that are recruited at specific stages to optimize the folding process and monitor the integrity of the protein product (Ellgaard et al, 1999; Hebert and Molinari, 2007). Encoded in the composition of N-linked glycans is information about the foldedness of the modified protein (Helenius and Aebi, 2004; Hebert et al, 2005). They serve as cellular maturation and quality control tags by recruiting, in a carbohydrate composition–dependent manner, a variety of factors that assist and guide the nascent chain through the early secretory pathway. A central player in the quality control decision in the ER is GT1 (UDP-glucose: glycoprotein glucosyltransferase; Trombetta and Parodi, 2003). Though little is known about its activity in its native biological environment, in vitro studies support the hypothesis that GT1 is the key quality control decision maker in the ER
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