Research

Type IV pilin signal peptides in S. solfataricus

An analysis of the recently published genome of Sulfolobus solfataricus showed that 4.2% of its proteome contains signal peptides. Most of these proteins are putative substrates for Sec-dependent secretion, but it appears that 10 proteins contain signal peptides homologues to bacterial type IV pilin signal peptides. Interestingly, this group of proteins not only comprises the flagellin gene as in other archaea, but includes mostly substrate-binding proteins associated with ABC (ATP-binding cassette) transport systems (Albers and Driessen, 2002). The type IV pilin cleavage site was confirmed by N-terminal sequencing three of the sugar-binding proteins (Elferink et al., 2001). In bacteria type IV pilin signal peptides are processed by a cytoplasmic signal peptidase such as pilD from Pseudomonas aeruginosa, which removes the signal peptide at the cytoplasmic side of the membrane and directly methylates the N-terminus of the mature pilin. Recently, in Methanococcus the first archaeal type IV pilin peptidase has been shown to process preflagellins. We identified ORF SSO0131 as the type IV pilin peptidase of S. solfataricus. In in vitro assays we could show that this enzyme, PibD, processes both the flagellin and the glucose-binding protein (Albers, Szabó and Driessen, 2003). PibD turned out to be a aspartyl signalpeptidase (Szabó, Albers and Driessen, 2006). 

 A detailed analysis of all available archaeal genomes showed that there are many proteins present which contain type IV pilin like (class III) signal peptides. A new type of prepilin peptidases was identified in Methanococcus marapaludis that processes specific proteins (Szabó et al, 2007), but the structure formed by these pilins is not yet identified.

  
 

Transport in Sulfolobus solfataricus

S. solfataricus grows optimally at a temperature of 80^oC and a pH of 2.5-3.5 on carbon sources such as yeast extracts, tryptone and various sugars. Glucose, arabinose, cellobiose, maltose and trehalose are taken up by binding-protein dependent ABC transporters. The binding proteins were purified, N-terminally sequenced and their genes identified in the S. solfataricus database. All these binding proteins are very pH stable, glycosylated, membrane-bound and bind their substrates with high affinities (KD<500 nM). The transporters can be divided into two categories. The first group is homologous to sugar transporters that contain one ATPase homodimer and a binding protein exhibiting an unusual signal sequence, which is similar to the bacterial IV pilin leader peptide sequence. The second group belongs to the family of oligo/dipeptide transporters that contain one ATPase heterodimer and a binding protein that has a typical bacterial signal sequence. However, S. solfataricus seems to prefer ABC type transport systems for sugar uptake (Albers et al., 1999) (Elferink et al., 2001).

The glucose transporter has been characterized in more detail. The genes have been cloned from the S. solfataricus P2 genome and expressed in E. coli. Expression of the membrane components is very low and could be achieved only by co-expression of tRNA genes, which are rare for E.coli, but frequently, used by S. solfataricus. The recombinant ATPase, GlcV, shows ATP hydrolysing activity and was biochemically characterized. The ATPase has been crystallized by G. Verdon and A. Thunnissen (Biophysics, University of Groningen). It shows two domains: an ATP hydrolyzing domain and a regulatory domain (see picture).