Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain.
We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with its location as an integral membrane protein, whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide. These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain. Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.
Shailubhai
K
K
Department of Animal Sciences, University of Maryland, College Park 20742.
Pukazhenthi
B S
BS
Saxena
E S
ES
Varma
G M
GM
Vijay
I K
IK
D000595:Amino Acid Sequence; D000818:Animals; D001940:Breast / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005033:Ethylmaleimide; D007425:Intracellular Membranes / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D014357:Trypsin; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism*
D008565:Membrane Proteins; C042059:glucosidase I; D000520:alpha-Glucosidases; D014357:Trypsin; D005033:Ethylmaleimide