Abstract
The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase beta isoforms expressed in vitro and in Xenopus oocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins.
Highlights
Introduction of Novel Glycosylation SitesClose to the  Transmembrane Domain Impedes Efficient ␣- Interaction and/or Intracellular Routing—our studies on the Na,K-ATPase  subunits have provided information on the location of putative sites of interaction with the partner ␣ subunit
For the glycosylation mapping assay, we have introduced Asn-Ser-Thr glycosylation acceptor sites at various positions around the predicted C-terminal ends of 1 and 3 subunit transmembrane domains and have used the concept of minimal glycosylation distance defined as the number of amino acids separating the C-terminal end of the transmembrane domain from the first Asn residue that is halfmaximally glycosylated [17]
Because half-maximal glycosylation occurs at a distance of about 10 –11 amino acid residues away from the C-terminal end of natural, synthetic, or heterologous transmembrane domains introduced into the model membrane protein leader peptidase (Lep) [17, 18, 33], our results suggest that the 1 transmembrane domain is shorter than predicted by KyteDoolittle hydropathy analysis and ends around Leu58
Summary
Introduction of Novel Glycosylation SitesClose to the  Transmembrane Domain Impedes Efficient ␣- Interaction and/or Intracellular Routing—our studies on the Na,K-ATPase  subunits have provided information on the location of putative sites of interaction with the partner ␣ subunit. Similar defects have previously been observed in vesicular stomatitis virus G proteins [46, 47] and influenza virus hemagglutinin [48] containing sugar chains at new glycosylation sites, and it was concluded from these studies that the primary role of natural sugars is to promote proper protein folding Neither in those nor in the present study, can it be definitively decided whether the alterations in the folding of the glycosylation mutants is due to the presence of a new sugar chain or to the amino acid substitutions introduced to create a new glycosylation site. Further mutational analysis is needed to determine whether the decreased ␣-interaction efficiency of the -glycosylation mutants reflects a discrete disruption of an assembly domain or is due to more general conformational perturbations introduced by the mutation and/or the sugar chain
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