A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition.

Albright, Charles F.; Orlean, Peter; Robbins, Phillips W.

doi:10.1073/pnas.86.19.7366

Cited by 90 publications

(40 citation statements)

References 31 publications

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“…Beyond catalysis, it is likely that this protein contains some specificity for the lipid portion of its substrate. Previous studies have identified a potential isoprenol recognition sequence for dolichol (FI/VxF/YxxIPFxF/Y) which we were unable to identify in BacA/UppP (Albright et al, 1989;Datta & Lehrman, 1993). We therefore hypothesize that the residues exhibiting identity in the first transmembrane domain constitute an undecaprenol recognition motif.…”

Section: Baca/uppp: the Principal Undecaprenyl Pyrophosphate Phosphatmentioning

confidence: 48%

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

Bickford

Nick

2013

Microbiology

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Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substratespecific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

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Section: Baca/uppp: the Principal Undecaprenyl Pyrophosphate Phosphatmentioning

confidence: 48%

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

Bickford

Nick

2013

Microbiology

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“…Thus, it is highly possible that those lysine residues that are proximal or within the transmembrane domains of OT subunits are involved in cross-linking among them. Indeed, this idea was further supported by the cross-linking of OT subunit Ost1p Ost1p with another ER membrane protein, Alg1p, with regard to their relationship as follows: several OT subunits (Ost1p, Wbp1p, Swp1p) were found containing the consensus sequence, which is distinctive among dolichol binding enzymes in their membrane-spanning domains and has been proposed to bind to the assembled lipid-linked oligosaccharides (25,26), and Alg1p was recently found to exist as a homo-oligomer and perform dual functions in sugar transfer and in the recognition of dolichol phosphate-derived substrates. 2 Therefore, Alg1p could serve as a good model to investigate our hypothesis if it ideally interacts with one or more OT subunits that are involved in recognition and the binding of lipid-linked oligosaccharide donors.…”

Section: Selectivity Of the Cross-linker-as Demonstrated Inmentioning

confidence: 86%

New Findings on Interactions among the Yeast Oligosaccharyl Transferase Subunits Using a Chemical Cross-linker

Yan

Ahmed

Qi³

et al. 2003

Journal of Biological Chemistry

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At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of crosslinking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.Over the past decade it has been established that in both higher eukaryotes and yeast, the enzyme oligosaccharyl transferase (OT), 1 is a complex consisting of multiple, nonidentical membrane protein subunits residing in the endoplasmic reticulum (ER) (1). In the case of the yeast Saccharomyces cerevisiae, nine different subunits have been cloned and identified. Among them, the genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; OST4 gene is essential for growth of the cell at 37°C, but not at 25°C; Ost3p, Ost5p, Ost6p subunits are not essential for the viability of the cell, but are required for maximal activity of the enzyme complex in catalyzing N-glycosylation (for review, see Refs. 1 and 2). To understand how these nine subunits interact with each other, a genetic approach using the yeast two-hybrid screen was utilized, but it failed to show interactions between any of the luminal domains of the yeast OT subunits (3). Studies on the mechanism of enzyme catalysis have proposed that Wbp1p may contain a site for the binding of the lipidlinked oligosaccharide substrate (4), and Stt3p was shown to be directly involved in peptide substrate recognition and/or the catalytic glycosylation process (5). However, there is little detailed information on how these two subunits interact to catalyze the glycosylation reaction.Study of the structural interactions of membrane proteins of the ER offers a special challenge because of the hydrophobicity of the proteins and the special environ...

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“…3c). A potential dolichol recognition sequence located in transmembrane domains of several dolichol-utilizing enzymes has been proposed (30). However, in the ALG8 glucosyltransferase sequence no region with a high homology to the potential dolichol recognition sequence F (I/V)X(F/Y)XXIPFX(F/Y) (30,31) was detected.…”

Section: Discussionmentioning

confidence: 99%

“…A potential dolichol recognition sequence located in transmembrane domains of several dolichol-utilizing enzymes has been proposed (30). However, in the ALG8 glucosyltransferase sequence no region with a high homology to the potential dolichol recognition sequence F (I/V)X(F/Y)XXIPFX(F/Y) (30,31) was detected. Direct evidence for the involvement of this sequence in the recognition or binding of dolichol is not available, but mutational analysis of this potential dolichol recognition sequence in hamster GlcNAc-1-phosphate transferase revealed that this sequence is essential for enzymatic activity (32).…”

Section: Discussionmentioning

confidence: 99%

New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus.

Štagljar

Heesen²,

Aebi³

1994

Proc. Natl. Acad. Sci. U.S.A.

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Glc3ManGkcNAc2 is the preferred substrate of the ofigosaccharyltransferase of N-linked glycosylation of proteins, but nonglucosylated oligoacharides can be transferred to proteins in Saccharomyces cerevisiae. Mutations affecfting the addition ofthe three terminal glucose residues lead to accmulation of ManGlcNAc2 or Glc1ManGIcNAc2 in vivo but do not show any detectable growth defect. When these mutations were introduced into a strain with reduced o saharyltransferase activity (due to the wbpl-l mutation), a severe growth defect was observed: accumulation of suboptimal lipid-linked oligosaccharide and reduced oligosaccharyltransferase activity resulted in a severe underglycosylation ofsecreted proteins. This new synthetic phenotype made it possible to isolate the ALG8 locus, encoding a potential glucosyltransferase of the endoplasmic reticulum. The ALG8 protein is a 63.5-kDa hydrophobic protein that is not essential for the vegetative growth of yeast. However, the lack of this protein resulted in underglycosylation of secreted proteins.

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A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition.

Cited by 90 publications

References 31 publications

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

New Findings on Interactions among the Yeast Oligosaccharyl Transferase Subunits Using a Chemical Cross-linker

New phenotype of mutations deficient in glucosylation of the lipid-linked oligosaccharide: cloning of the ALG8 locus.

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