Two distinct domains of the beta-subunit of glucosidase II interact with the catalytic alpha-subunit.
Recent purification and cDNA cloning of the endoplasmic reticulum processing enzyme glucosidase II have revealed that it is composed of two soluble proteins: a catalytic alpha-subunit and a beta-subunit of unknown function, both of which are highly conserved in mammals. Since the beta-subunit, which contains a C-terminal His-Asp-Glu-Leu (HDEL) motif, may function to link the catalytic subunit to the KDEL receptor as a retrieval mechanism, we sought to map the regions of the mouse beta-subunit protein responsible for mediating the association with the alpha-subunit. By screening a panel of recombinant beta-subunit glutathione S-transferase fusion proteins for the ability to precipitate glucosidase II activity, we have identified two non-overlapping interaction domains (ID1 and ID2) within the beta-subunit. ID1 encompasses 118 amino acids at the N-terminus of the mature polypeptide, spanning the cysteine-rich element in this region. ID2, located near the C-terminus, is contained within amino acids 273-400, a region occupied in part by a stretch of acidic residues. Variable usage of 7 alternatively spliced amino acids within ID2 was found not to influence the association of the two sub-units. We theorize that the catalytic subunit of glucosidase II binds synergistically to ID1 and ID2, explaining the high associative stability of the enzyme complex.
Arendt
C W
CW
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
Ostergaard
H L
HL
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016399:Lymphoma, T-Cell; D051379:Mice; D018000:Receptors, Peptide / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
C064459:KDEL receptor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases