Abstract
Calnexin is an integral membrane protein that acts as a chaperone during glycoprotein folding in the endoplasmic reticulum. Cross-linking studies were carried out with the aim of investigating the interactions of calnexin with glycoproteins in vitro. A truncated version of the integral membrane glycoprotein Glut 1 (GT155) was synthesized in a rabbit reticulocyte translation system in the presence of canine pancreatic microsomes. Following immunoprecipitation with an anticalnexin antiserum, a cross-linker-independent association was observed between GT155 and calnexin. In addition, the anti-calnexin antiserum immunoprecipitated a UV-dependent cross-linking product consisting of GT155 and a protein of approximately 60 kDa designated CAP-60 (calnexin-associated protein of 60 kDa). Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions. A GT155 mutant that was not glycosylated (AGGT155) did not associate with calnexin or CAP-60. Calreticulin, the soluble homologue of calnexin, was also shown to interact with GT155 only when the protein bore a correctly modified oligosaccharide group. Thus, our data show that both calnexin and calreticulin with Glut 1 in a glycosylation-dependent manner.
Highlights
Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions
Further evidence that membrane insertion of GT155 occurred in this system was provided by the resistance of the polypeptide to protease digestion (Fig. 1A, lane 4 compared with lane 5)
The interaction between calnexin and nascent glycoproteins has been studied in various ways
Summary
Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions. Membrane and secretory proteins are co-translationally translocated into the endoplasmic reticulum (ER) membrane or lumen in an unfolded or partially folded state [1]. To ensure that these proteins reach their subcellular destination in the correct conformation a variety of chaperones are required [2]. This dissociation often correlates with the acquisition of “transport competence” by the newly synthesized protein, allowing it to exit the ER and be transported to later compartments of the secretory pathway In this way chaperones such as calnexin and BiP function to ensure “quality control” of proteins during transit through the ER [3, 4]
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