Identification of diverse archaeal proteins with class III signal peptides cleaved by distinct archaeal prepilin peptidases

Abstract

Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal prepilin-peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.

FIG. 1.

N-terminal signal peptide structures. Tripartite structure of class I (secretory) or class II (lipoprotein) signal peptides and class III (type IV pilin-like) signal peptide. Signal peptide cleavage by signal peptidases I and II, and prepilin peptidase, respectively, is symbolized by scissors; dark gray, hydrophobic region; light gray, cleavage region; +, positive charges.

FIG. 2.

Cleavage of euryarchaeal pilin-like proteins by a dedicated signal peptidase. (A) Schematic representation of the conserved M. maripaludis and M. jannaschii operons containing genes that encode proteins with DUF361-like domains (black) and the novel subclass of euryarchaeal prepilin peptidase, eppA (white). Homologous genes are highlighted by identical shading. Note that the operon is split in M. jannaschii. (B) Transmembrane topologies of FlaK (left) and EppA (right) from M. maripaludis, predicted using the Phobius web server (21). Homologous regions in EppA and FlaK are in gray. A predicted four-transmembrane insertion in EppA is indicated in white. (C) Signal peptide cleavage of preproteins containing Duf361-like domains (EpdA and EpdC) and flagellin (FlaB2) from M. maripaludis by EppA or FlaK, as indicated. Control, E. coli expressing preprotein in the absence of an archaeal prepilin peptidase. Genes encoding precursor proteins under the arabinose-inducible promoter were expressed alone or in combination with IPTG-inducible peptidase genes. (D) Signal peptide cleavage of EpdA and EpdC with the flagellin cleavage site sequence or FlaB2 with the EpdA cleavage site sequence by EppA or FlaK. p, precursor, m, mature.

FIG. 3.

Structural conservation of operons encoding pilin-like FlaFind positives and a helicase; schematic representation of Sulfolobus and Pyrococcus operon structures. Genes coded for proteins with predicted functions, including FlaFind-positive proteins (green); proteins with a DUF361-like domain (hatched); an Lhr-like helicase (purple); a cell division GTPase/FtsZ3 (turquoise); COG5306, an uncharacterized conserved protein (light gray); COG4025, a predicted membrane protein (black); a TadA/VirB11-like ATPase (red); a TadC-like membrane protein (brown); an EndoIII-related endonuclease (COG0117, magenta); a transcriptional regulator (COG1474, cyan); a glycosyltransferase probably involved in cell wall biogenesis (COG1215, pink); a protein conserved in Sulfolobus (dark gray); and proteins conserved in Pyrococcus (white).