AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7

Abstract

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D₁₀₀), IV (F₂₂₀) and V (F₂₆₄) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Keywords: AglH; Alg7; Crenarchaea; Dolichol phosphate; Glycosylation; N-Glycosylation; Sulfolobus.

Fig. 1

Physical map of the gene region adjacent to algH of S. acidocaldarius. Illustrated are the genes Saci0088 until Saci0096. The gene aglH displayed in black (saci0093, SACI_RS00435) encodes the UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase. The genes idi (saci0091) and gds (saci0092) are involved in the isoprenoid lipids biosynthesis

Fig. 2

Topology model of AglH from S. acidocaldarius. The topological model was derived using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) and PSIPRED protein structure prediction server (http://bioinf.cs.ucl.ac.uk/psipred/). Conserved amino acids (see alignment Fig. 3) are shown in bold. Numbers indicate internal cytoplasmic loops I to V, as well as their conserved motifs. Proposed catalytic interaction with UDP-GlcNAc is displayed in light grey. Boxed amino acids were replaced by alanine

Fig. 3

Alignment of archaeal, bacterial, and eukaryal UDP-GlcNAc-1-P transferase orthologs. Orthologs were identified using BLAST with S. acidocaldarius Saci0093 (AglH). Protein sequences were aligned with the ClustalW program. The partial alignment is shown for the region encompassing the conserved motifs DxxK (CL I), DDxxN/D (CL II) and NxxNxxxxGxxGxxxxG (CL III). Conserved amino acids are indicated with asterisks. The boundaries of transmembrane domains (filled boxes) and cytosolic loops (lines) are shown on top of the alignment. The replacement of selected amino acids from AglH of S. acidocaldarius by alanine (a) are indicated above the sequence

Fig. 4

Effect on the cell growth of S. acidocaldarius by the N-glycosylation inhibiting antibiotics bacitracin and tunicamycin. a Different concentrations of bacitracin: 0 mM (filled circle), 0.6 mM (filled triangle), 1.2 mM (filled diamond), 2.4 mM (filled square) mM were added after reaching exponential phase, indicated by black arrow. b Different concentrations of tunicamycin: 0 (filled circle), 2 µg/ml (filled triangle), 6 µg/ml (filled diamond), 16 µg/ml (filled square), 32 µg/ml (open square) were added after reaching exponential phase. c Cell growth without antibiotics (filled circle), with 1.2 mM bacitracin (filled square), or 6 µg/ml tunicamycin (filled triangle), antibiotics were added directly after inoculation of the culture, indicated by black arrow

Fig. 5

Rescue of a conditional lethal alg7 yeast mutant by the thermophilic aglH from S. acidocaldarius. YPH499-HIS-GALprom-ALG7 was transformed with the pRS426-METt plasmids carrying either the human ALG7 (Hs-alg7) or the archaeal Saci0093 (Saci-aglH). Transformed cells were streaked onto plates containing minimal medium lacking histidine and uracil and containing either galactose or glucose

Fig. 6

Functional characterization of AglH in YPH499-HIS-GAL-ALG7 by a complementation assay. The conditional lethal mutant YPH499-HIS-GAL-ALG7 was transformed with the pRS426-METt plasmid carrying either the archaeal aglH (Saci0093) or aglH with selected point mutations, resulting in the exchange of conserved amino acids to alanine. The transformed cells were then streaked onto plates containing minimal medium lacking histidine as well as uracil and containing either galactose (a) or glucose (b). The exchange of the conserved amino acids D₁₀₀A, F₂₆₄A resulted in a lethal, whereas the exchange of F₂₂₀A resulted in a slow-growing phenotype of YPH499-HIS-GAL, under repression condition of the alg7 gene (b)

Fig. 7

Detection of the protein expression from the derived AglH_FLAG point mutation by Western immunoblotting. Equivalent amounts of cells from YPH499 background strain (lane 1) or the complemented strains with the AglH_FLAG expression vector (lane 2–9) were separated by 11% SDS-PAGE and immunoblotted with antibodies raised against the FLAG-tag epitope. The non-mutated AglH_FLAG (n. m.) showed a similar expression level as the different AglH_FLAG point mutations (lane 3–9)