Yeast GTB1 Encodes a Subunit of Glucosidase II Required for Glycoprotein Processing in the Endoplasmic Reticulum

Abstract

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose ␣-subunit contains the glycosyl hydrolase active site. The function of the ␤-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the ␤-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96 -102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast ␣-subunit homologue Gls2p. Surprisingly, we found that ⌬gtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…This is a heterodimer of catalytic Gls2/Rot2 and Gtb1, the latter of which is necessary for, and influences the rate of, Glc trimming (Trombetta et al 1996;Wilkinson et al, 2006;Quinn et al 2009).…”

“…Processing proceeds by removal of the a1,2-linked Glc by glucosidase I, Gls1/Cwh41 (Romero et al 1997), and then of the two a1,3-linked Glc by soluble glucosidase II, a heterodimer of catalytic Gls2/Rot2 and Gtb1 (Trombetta et al 1996;Wilkinson et al 2006;Quinn et al 2009; Figure 2). ER mannosidase I, Mns1, removes an a1,2 Man to generate GlcNAc 2 Man 8 Herscovics 1999), and, if correctly folded, proteins bearing this glycan are exported from the ER.…”

Deficiency of hepatocystin induces autophagy through an mTOR-dependent pathway

“…The removal of glucose residues by Glu II is critical in the processing and folding of glycoproteins in the ER. [10][11][12] Since hepatocystin knockdown impaired Glu II activity ( Fig. 2A), we reasoned that it might induce autophagy through disturbing the UPR pathway.…”

“…[5][6][7][8][9] The heterodimeric Glu II complex is responsible for the removal of the two innermost glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. 10,11 These two cleavage events are important steps during nascent glycoproteins' entry and exit from calnexin/ calreticulin-assisted folding process. 12 In addition to its role in glycoprotein folding, hepatocystin has recently been shown to interact with and regulate the inositol-1,4,5-trisphosphate receptor (IP3R), an intracellular signaling hub on the ER.…”

Deciphering the Roles of Glycan Processing in Glycoprotein Quality Control through Organic Synthesis

“…G-II is a dual activity enzyme, successively cleaving the Glc1-3Glc (cleavage-1) and Glc1-3Man (cleavage-2) linkages [52][53][54][55] (Fig. 1A).…”