Alg14 Recruits Alg13 to the Cytoplasmic Face of the Endoplasmic Reticulum to Form a Novel Bipartite UDP-N-acetylglucosamine Transferase Required for the Second Step of N-Linked Glycosylation

Abstract

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc 2 -PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc 2 -PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.Asparagine (N)-glycosylation is an essential modification that regulates protein folding and stability. Prior to its attachment to protein, the oligosaccharide Glu 3 Man 9 GlcNAc 2 is assembled on the lipid carrier, dolichyl pyrophosphate (dol-PP), in the ER 2 (see Refs. 1-4 for review). The earliest steps of this lipid-linked oligosaccharide (LLO) synthesis begin on the cytoplasmic face of the ER. Seven sugars, (two N-acetylglucosamines and five mannoses) are sequentially added to dol-P to form Man 5 GlcNAc 2 -PP-dol by enzymes that have their catalytic domain on the cytosolic side of the ER membrane and use sugar nucleotide substrates (2, 5, 6). The enzymes that catalyze addition of the next seven sugars (four mannoses and three glucoses) do so within the lumen of the ER and use dolichol-linked sugar substrates (see Ref. 1 for review). Once assembled, this core oligosaccharide is transferred to protein by oligosaccharyltransferase through an N-glycosidic bond to an asparagine that is part of the Asn-X-(Ser/Thr) consensus sequence (7). Proteinlinked oligosaccharide is immediately modified by the removal of glucoses and mannose by ER glucosidases and mannosidases. Failure to properly synthesize, transfer, or modify the N-linked glycan results in glycoproteins that are recognized by the quality control systems that restrict these abe...

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

“…3A) agrees well with previous reports in which it was demonstrated that they associate with each other with high affinity to form a tight complex, using co-immuno-precipitation assays by ourselves (18) and pull-down assays by another group (19 (HIS3 and ADE2). Co-transformant cells expressing both bait and prey proteins were selected on synthetic dextrose (SD) minimal medium lacking leucine and tryptophan (SD-LW), then transferred onto SD medium lacking leucine, tryptophan, histidine (SD-LWH), and to SD medium lacking leucine, tryptophan, histidine, and adenine (SD-LWHA), and incubated at 30 ˚C for 3 days.…”

Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum

“…Plates were incubated for 4 days at 24°C. NPC biogenesis and lipid homeostasis luminal marker Kar2-mRFP-HDEL (Gao et al, 2005). Endogenously produced sterols showed similar localization, as revealed by staining with filipin (data not shown).…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…These proteins function as a heterodimer to transfer the second, β1,4--GlcNAc--linked GlcNAc to Dol--PP--GlcNAc (Bickel et al 2005;Chantret et al 2005;Gao et al 2005). Soluble Alg13, assigned to GT Family 1, is the catalytic subunit and associates with membrane--spanning Alg14 at the cytosolic face of the ER membranes (Averbeck et al 2007;).…”

“…Assembly and transfer of the Dol-PP-linked precursor oligosaccharide: Steps on the cytoplasmic face of the ER membrane: These steps are (i) transfer of GlcNAc-1-P from UDP-GlcNAc to Dol-P by Alg7, the target of the N-glycosylation inhibitor tunicamycin (Barnes et al 1984), (ii) transfer of b1,4-GlcNAc from UDP-GlcNAc by heterodimeric Alg13/Alg14 (Bickel et al 2005;Chantret et al 2005;Gao et al 2005), (iii) transfer of a b1,4-linked Man by Alg1 (Couto et al 1984), (iv) successive transfer of an a1,3 and an a1,6 Man by Alg2 (O'Reilly et al 2006;Kämpf et al 2009), and (v) transfer of two a1,2-linked Man by Alg11 (Cipollo et al 2001;O'Reilly et al 2006;Absmanner et al 2010). These proteins act in higher-order complexes Noffz et al 2009;File S2).…”