AglH, a predicted UDP-GlcNAc-1-phosphate: dolichyl phosphate GlcNAc-1-phosphotransferase, is initi-ating the protein N-glycosylation pathway in the thermoac-idophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phos-photransferase activity of Alg7 in a conditional lethal Sac-charomyces 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 Methanococ-cus voltae (Shams-Eldin et al. 2008). The topology predic-tion 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 cytoplas-mic 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-phosphotrans-ferases conﬁrmed the essentiality of N-glycosylation for cell survival.
|Number of pages||14|
|Early online date||7 Nov 2016|
|Publication status||Published - Jan 2017|
|Event||11th International Congress on Extremophiles - Kyoto, Japan|
Duration: 12 Sep 2016 → 16 Sep 2016
- Dolichol phosphate