Organization and interactions of cell envelope proteins of the extreme thermoacidophileSulfolobus acidocaldarius

Abstract

To address basic questions concerning proteins of the Suljolobus acidocaldarius cell1 envelope, cell ghosts (empty cells consisting of cytoplasmic membrane complexed with the glycoprotein S-layer) were iso1;xted and analyzed by standard biochemical methods. The constituent proteins included the two dissimilar subunits also founcl in the baglike structures (sacculi) of S-layer (purified from whole cells by detergent extraction), plus approximately 30 additional protein species. When S-layer-dissociating conditions were applied to intact cell ghosts, the large sacculus subunit was released, whereas the small subunit remained bound to the cytoplasmic membrane. These results provided evidence that the small subunit may anchor the S-layer to the S. acidocaldarius cell membrane. The S. acidocaldarius S-layer was highly resistant to proteolysis in its native state but not when dissociated into subunits. Virtually all other membrane-associated proteins were readily digested by pronase treatment of cell ghosts. Pronase treatment of whole cells, in contrast, removed only a defined subset of the membrane-associated proteins, suggesting that this subset represents a few proteins nonnally exposed to the external medium. Several putative membrane glycoproteins were detected in cell ghosts that were not demonstrably associated with the S-layer.Resume : Pour rkpondre B certaines questions fondamentales sur les protkines de l'enveloppe cellulaire du Suljolobus acidocaldarius , nous avons is016 des ccfant6mes~ de cellules (cellules vides comprenant la membrane cytoplasmique complex6e avec la glycoprotkine de la couche S) et les avons analysks par les mkthodes biochiimiques usuelles. Les protkines constituantes comprennent deux sous-unit6s non-semblables aussi retrouvkes dans des structuires en forme de sac (saccules) de la couche S (purifike B partir de cellules entibres par extraction avec des dktergents), ainsi qu'environ 30 autres sortes de prot6ines. Lorsque les fant6mes cellulaires sont placCs dans des conditions permettant de dissocier la couche S, la grosse sous-unit6 du saccule est detachke, mais la petite sous-unit6 demeure like B la membrane cytoplasmique. Ces rksultats confirment que la petite sous-unit6 peut accrocher la couche S B la membrane cellulaire du S. izcidocaldarius. La couche S du S. acidocaldarius est formement rksistante B la protColyse B 1'Ctat nature1 mais pas lorsqu'elle est scind6e en sous-unit6s. Toutes les autres protkines membranaires sont rapidement digkrkes par un traitment 2 la pronase des fant6mes de cellules. Un m&me traitement sur des cellules entibres n'enlbve qu'une portion prkcise de l'assemblage des protkines associkes 2 la membrane suggCrant que cette portion ne reprksente que quelques protkines normalement exposkes au milieu exteme. Quelques glycoprotkines membranaires putatives ont Ctk dktect6es dans les fant6mes de cellules, mais leur association avec la couche S n'a pas 6t6 dkmontrke.

Posttranslational Protein Modification inArchaea

“…Relying on glyco-staining, lectin-binding techniques, and treatments with inhibitors of glycosylation or deglycosylating agents, the membranes of both Sulfolobus acidcaldarius and Sulfolobus solfataricus were shown to contain unidentified glycoproteins distinct from the S-layer glycoprotein (147,262). Glycoprotein staining was used to identify a series of glycosylated proteins in Pyrococcus furiosus membranes that are distinct from CipA and CipB and the expression of which is related to growth temperature (464).…”

“…Tunicamycin hinders transfer of UDP-Nacetylglucosamine to polysaccharide-loaded dolichol carriers (105). Treatment with this antibiotic interferes with Sulfolobus acidocaldarius S-layer glycoprotein glycosylation (147). In contrast, tunicamycin has no effect on the biosynthesis of the Haloferax volcanii S-layer glycoprotein (99) and accordingly, the glycan moiety of the Haloferax volcanii S-layer glycoprotein does not include N-acetylglucosamine (242,280).…”

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.…”

Protein glycosylation in Haloferax volcanii: partial characterization of a 98-kDa glycoprotein

“…Apart from S-layer glycoproteins and £agellins, only a limited number of other glycosylated archaeal proteins have been reported [2,3], such as the membrane-associated glycoprotein of Thermoplasma acidophilum [12] and a series of glycoproteins from isolated membranes of Sulfolobus acidocaldarius [13]. Glycosylation of archaeal extracytoplasmic proteins may, however, be more widespread than currently thought.…”