Primary structure and glycosylation of the S-layer protein of Haloferax volcanii

Abstract

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanit) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N-and 0-glycosidic bonds. Glucosyl-(l-*2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549EMBO J. 7: -1554EMBO J. 7: , 1988.

Insights into ABC Transport in Archaea

“…Binding proteins belonging to the CUT class exhibit an N-terminal signal peptide often followed by a long stretch of hydroxylated amino acids up to 60 residues long, which might act as a flexible linker region between the membrane and the binding domain. A similar site in the S-layer of Halobacteria also has been shown to be highly glycosylated (Sumper et al, 1990). Binding proteins of archaea need to be attached to the cytoplasmic membrane as these organisms lack an outer membrane, whereas the S-layer is permeable for small proteins.…”

“…A common feature of all these binding proteins is their glycosylation. Glycosylation was believed to be absent in prokaryotes, but most studied extracellular proteins of archaea have been shown to be glycosylated (Erra-Pujada et al, 1999;Greller et al, 2001;Hettmann et al, 1998;Sumper et al, 1990). Binding proteins isolated from P. furiosus contain glucose moieties (Koning et al, 2002), whereas mannose, glucose, galactose, and N -acetylglucosamine have been identified in binding proteins of S. solfataricus (Elferink et al, 2001).…”

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

“…Such differences could affect the relative ease of obtaining IMVs from the haloarchaeal species. Furthermore, while the H. salinarum and H. volcanii S-layer glycoproteins are believed to be directly anchored into the plasma membrane [27,39], additional proteins are proposed to be responsible for the anchoring of the S-layer glycoprotein in Sulfolobus [1,18]. Although it is not known how the presence of rigid S-layers affects archaeal membrane lipid behavior [13], the suggested differences in haloarchaeal and Sulfolobus S-layer glycoprotein attachment could affect the ability of the membranes to reseal in an inverted manner.…”

“…The H. volcanii S-layer glycoprotein is found on the external face of the plasma membrane, apparently linked to the membrane via a stretch of hydrophobic amino-acid residues located near the C-terminus of the protein [39]. In vesicles of inverted topology, i.e., IMVs, the S-layer glycoprotein would face the vesicle interior and hence be inaccessible to externally added protease.…”

“…Failure to obtain IMVs from the vast majority of archaeal species in which vesicle preparation was attempted is generally attributed to the presence of a proteinaceous surface (S)-layer, a rigid structure often intimately associated with the plasma membrane [36]. Despite the presence of a protein-based shell comprised of a single glycoprotein species [25,39], we report successful preparation of large-scale amounts of IMVs from the halophilic archaeon Haloferax volcanii. In the following, these vesicles are characterized in terms of membrane orientation, sealing and functional behavior.…”