Arachaebacteria have been recently placed in evolution as a separate kingdom of 
organisms between procaryotes and eucaryotes. Although these organisms contain 
both glycolipids and glycoproteins, they possess no Golgi. No biosynthetic work 
has been published on the complex carbohydrates of these newly reassigned 
organisms. This report describes preliminary results from one member of this 
kingdom, Haloferax volcanii, which suggest that all glycosylation proceeds 
through lipid intermediates. Evidence for novel glycolipid structure was also 
found during this study. H. volcanii plasma membranes contain all of the enzyme 
activities for synthesis of N-linked glycoproteins and archaeol-based 
glycolipids. For glucose transfer, all reactions apparently proceed through 
glucose-phosphopolyisoprenol using UDP-glucose as primary donor. Incorporation 
of D-[3H]glucose from UDP-D-[3H]glucose into glycoproteins and glycolipids of H. 
volcanii was stimulated by addition of C55-polyisoprenol phosphate, but not by 
C85-105 dolichol phosphate, and was inhibited by amphomycin and two recently 
described sugar nucleotide analogs, PP36 
(5'-[N-(2-decanoylamino-3-hydroxy-3-phenylpropyloxy 
carbonyl)glycyl]amino]-5'-deoxyuridine) and PP55 
(5'-O-[[(2-decanoylamino-3-phenylpropyloxycarbonyl) amino]sulfonyl]uridine). All 
three inhibitors are reported to block transfer of sugar from UDP-sugars to 
phosphopolyisoprenols in eucaryotes. However, in H. volcanii these inhibitors 
apparently block transfer of glucose from polyprenyl intermediates to final 
glycoproteins and glycolipid products. The sulfodihexosyl archaeol glycolipid 
fraction was partially characterized by mass spectrometry and was found to 
contain a previously unreported structure with sulfate on the reducing-end 
sugar. Four major glycoproteins 190, 105, 56, and 52 kDa and an archaeol-based 
glycolipid fraction were labeled by amphomycin-sensitive pathways. Photoaffinity 
labeling of H. volcanii homogenate with 5-azido-[32P]UDP-Glc tagged only one 
45-kDa polypeptide which is a probable glucosyl-phosphorylpolyisoprenol 
synthase. The fact that only one polypeptide band was photoaffinity-labeled 
indicated that no other transferase utilized UDP-glucose directly in H. 
volcanii. The salt requirement of the UDP-glucose-dependent pathways suggests 
that cytoplasmic enzymes function in a high salt environment in H. volcanii. The 
archaebacterial plasma membrane thus expresses many functions for glycosylation 
of both glycoproteins and glycolipids, normally found in the endoplasmic 
reticulum and Golgi of eucaryotes.