In a recent MicroCorrespondence, Albers et al. (1999, Mol Microbiol31: 1595–1596) proposed that membrane-anchored secretory proteins in archaea are processed and translocated by a mechanism similar to that of archaeal flagellins, the major structural components of the archaeal flagellum. Work in the last decade has clearly identified the archaeal flagellum as a unique motility apparatus (Jarrell et al., 1996, J Bacteriol178: 5057–5064; Bayley and Jarrell, 1998, J Mol Evol.46: 370–373). For example, analysis of completely sequenced genomes of several flagellated archaea has failed to reveal homologues to any genes that encode structural components of bacterial flagella (hook, rings, rod, filament, hook-associated proteins or motor), indicating that the archaeal motility structure is distinct in both composition and assembly (Faguy and Jarrell, 1999, Microbiology145: 279–281). Additionally, a fascinating discovery was that archaeal flagellins, unlike their bacterial counterparts, are made with an unusual 11- or 12-amino-acid leader peptide that is absent in the flagellins isolated from flagellar filaments (Kalmokoff and Jarrell, 1991, J Bacteriol173: 7113–7125; Bayley et al., 1998, Mol Gen Genet258: 639–645).
Archaeal flagellins do not show homology to bacterial flagellins, but do possess similarities to the N-termini of pilins of the type IV pilus (Faguy et al., 1994, Can J Microbiol40: 67–71), a bacterial structure involved in adhesion but perhaps primarily in twitching motility (Mattick and Alm, 1995, Trends Microbiol Sci3: 411–413). Type IV pilins have an unusual, short leader peptide that is processed by an enzyme called the prepilin peptidase. This bifunctional enzyme, PilD in Pseudomonas aeruginosa (Nunn and Lory, 1991, Proc Natl Acad Sci USA88: 3281–3285), cleaves after an invariant glycine and methylates the resulting N-terminal phenylalanine residue. The −2 and −3 positions in the case of the prepilin are usually lysine and glutamine respectively, the +5 position is glutamic acid and the N-terminus of the mature pilin is extremely hydrophobic. Similarly, methanogen preflagellins (flagellins with attached leader peptides), and probably all archaeal preflagellins, are also processed after an invariant glycine and the −2 is always a charged amino acid, often lysine as in prepilins. The −3 position is also always a charged amino acid. All archaeal flagellins possess a very hydrophobic N-terminus and some archaeal flagellins [A. fulgidus and Methanospirillum hungatei (Faguy et al., 1994, J Bacteriol176: 7491–7498)] also possess the +1 phenylalanine and +5 glutamic acid typical of type IV pilins. Searches of the completely sequenced genomes of flagellated archaea have not revealed homologues to prepilin peptidases.
The prepilin peptidase is also responsible for the processing of several other substrates (termed pseudopilins) which are necessary for the secretion of proteins in Gram-negative bacteria via the general secretory pathway (Nunn and Lory, 1992, Proc Natl Acad Sci USA89: 47–51; Filloux et al., 1998, FEMS Microbiol Rev, 22: 177–198). The pseudopilins, in general, share a number of the conserved features of type IV pilins including short positively charged leader peptides, the −1 glycine, the +5 glutamic acid and the highly hydrophobic N-terminal domain. The −2 and −3 positions are usually lysine and glutamine like the pilins, whereas the +1 phenylalanine position is often different.
Recent additional evidence further supports a relationship between archaeal flagella and type IV pili (Bayley and Jarrell, 1998, J Mol Evol.46: 370–373). In close proximity to the flagellins in all archaeal cases examined (Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, Methanococcus jannaschii, Pyrococcus abyssi, Pyrococcus horikoshii, Archaeoglobus fulgidus, Aeropyrum pernix), lies a gene with strong homology to pilB/T, which encodes an ATPase in the type IV pilus system. This protein may provide the motor for the twitching motility associated with type IV pili (Wall and Kaiser, 1999, Mol Microbiol32: 1–10) and for flagella rotation in the archaea.
From our work on methanogens (M. voltae, M. vannielii, M. maripaludis, M. thermolithotrophicus) and the completely sequenced archaeal genomes (M. jannaschii, P. abyssi, P. horikoshii, A. fulgidus, A. pernix), it appears that all archaea possess multiple tandem flagellin genes. Leader peptides have been identified directly in the cases of M. voltae and M. vannielii by comparing N-terminal sequences of purified flagellin proteins with the gene sequences (Kalmokoff and Jarrell, 1991, J Bacteriol173: 7113–7125; Bayley et al., 1998, Mol Gen Genet258: 639–645). Recently, we have also developed an in vitro assay for the preflagellin peptidase activity responsible for cleavage of the leader peptides from the preflagellins of M. voltae. In this assay, M. voltae preflagellin subunits overexpressed in Escherichia coli (substrate) were mixed with methanogen membranes (enzyme source) and the appearance of mature flagellin was observed by immunoblotting using anti-flagellin sera (Correia and Jarrell, submitted manuscript). With this assay, we have detected preflagellin peptidase activity in a variety of methanococci. In the other archaea listed above, leader peptides can be putatively identified. The lengths range from four to 15 amino acids, with most in the range of 11–12. In every case (34 different flagellins from 10 organisms representing mesophiles, thermophiles, hyperthermophiles, halophiles, methanogens and including members of both kingdoms of archaea, the Euryarchaeota and the Crenarchaeota), the conservation of amino acid sequence around the cleavage site of the leader peptide is striking (Table 1). The −1 position is always glycine. The −2 and −3 positions are always a charged amino acid, usually lysine or arginine, but sometimes the −3 position is glutamic acid (H. halobium and A. fulgidus). In some cases, the predicted leader peptide consists of little more than the KKG/RRG motif (as in P. horikoshii and P. abyssi). We suggest that the maintenance of these conserved sequences throughout such a diverse representation of archaea, inhabiting a variety of extreme environments, may mean that this sequence is required for proper recognition by, or activity of, the preflagellin peptidase.
Albers et al. (1999, Mol Microbiol31: 1595–1596) have suggested that various membrane-anchored secretory proteins may also be processed by the same enzyme that process the archaeal flagellins. The potential substrates suggested by Albers et al. (1999, Mol Microbiol31: 1595–1596), via searches of archaeal gene banks, include cutinases, amylases, the S layer protein of M. jannaschii and sugar-binding proteins that have predicted short atypical leader peptides. Included as well is the glucose binding protein (GBP) of Sulfolobus solfataricus, which is the only one with a demonstrated leader peptide ending in glycine. The proposed substrates have a positively charged amino terminus corresponding to a predicted leader peptide and a stretch of hydrophobic amino acids after the glycine, as found in both archaeal flagellins and type IV pilins.
However, a closer examination of the potential substrates, and in particular the leader peptides, indicates that although lysine and arginine residues are often present, they are not usually present in the −2 and −3 positions, and never in both positions in the same leader peptide. Since the −2 and −3 positions are always held by charged amino acids in archaeal preflagellins, no matter what the source, we feel that it is unlikely that the preflagellin peptidase will also be the same enzyme that processes all the membrane-bound secretory proteins as well.
If we examine the case of the bacterial prepilin peptidase, we see that the natural substrates have a very conserved amino acid sequence surrounding the cleavage site, even though mutant studies indicate that these conserved amino acids, other than the −1 glycine, are not required. Except for the −1 glycine, single and multiple amino acid substitutions in the leader peptide and amino-terminal conserved region of the type IV pilin from P. aeruginosa generally had a surprising lack of effect on subsequent processing by the prepilin peptidase (Strom and Lory, 1991, J Biol Chem266: 1656–1664; Strom and Lory, 1992, J Bacteriol174: 7345–7351; MacDonald et al., 1993, Can J Microbiol39: 500–505). Substitution of the prepilin −1 glycine with almost any other amino acid inhibited cleavage of the leader peptide and prevented assembly of the pilus. Only alanine in the place of glycine allowed even partial processing of prepilin (Strom and Lory, 1991, J Biol Chem266: 1656–1664). Substitutions for the conserved −2 lysine, as well as several of the other residues in the short leader peptide, did not affect subsequent proper processing by the prepilin peptidase. Furthermore, substitutions of the +1 phenylalanine with either a polar, hydrophobic or charged residue did not affect the post-translational processing of the prepilin and neither did the majority of other amino acid substitutions in the highly conserved N-terminal region of the pilin. Despite these allowances in sequence, there are relatively few natural substrates (pilin and pseudopilin precursors) that have substitutions in the −3 to +1 and +5 positions. In addition, despite the sequence similarity of archaeal preflagellins and type IV prepilins around the cleavage site, preflagellins of M. voltae are not processed by the P. aeruginosa prepilin peptidase (Bayley and Jarrell, 1999, J Bacteriol181: 4146–4153).
In M. voltae, the only other demonstrated protein with a leader peptide is the S layer protein, initially reported as an ATPase (Dharmavaram et al., 1991, J Bacteriol173: 2131–2133). This 12-amino-acid-long leader peptide is cleaved after an alanine and has no charged amino acids. In addition, the leader peptide is not followed by a stretch of hydrophobic amino acids, but instead has acidic or basic amino acids in eight of the first 21 positions of the mature protein. It seems that this protein would be a very unlikely candidate for processing by the same enzyme that cleaves the M. voltae flagellins. It also seems unlikely that a single enzyme in M. jannaschii would process the flagellins and the S layer protein (a candidate listed by Albers et al., 1999, Mol Microbiol31: 1595–1596), but different enzymes would perform this function in its relative M. voltae. Also noteworthy are the S layer proteins of Methanothermus sociabilis and Methanothermus fervidus, flagellated hyperthermophilic methanogens, in which typical, bacterial-like, N-terminal leader peptides of 22 amino acids are predicted with cleavage following a alanine–glycine–alanine (Bröckl et al., 1991, Eur J Biochem199: 147–152). As in the S layer protein of M. voltae, the amino acid sequences following the predicted cleavage site in the S layer proteins of both M. fervidus and M. sociabilis are not hydrophobic, but instead contain numerous acidic and basic residues. Assuming the flagellins of these methanogens have the same conserved amino acids surrounding the cleavage site as observed in all other archaea, it seems unlikely that the same enzyme would process both the flagellins and the S layer proteins in these organisms either.
We are currently generating preflagellin mutant species that have single amino acid changes in the −1 to −3 positions to test their susceptibility to cleavage in the in vitro assay. This should indicate whether the −1 glycine and the −2 and −3 charged amino acids are absolutely required for cleavage by the preflagellin peptidase. The availability of this assay for preflagellin peptidase activity should allow for the identification of the gene encoding the enzyme responsible for this function in M. voltae. We believe that the preflagellin peptidase will prove to be a more or less dedicated enzyme for the processing of the preflagellins and perhaps a limited number of related proteins, much as the prepilin peptidase recognizes only prepilin and pseudopilin substrates. However, until experimental data are available the proposal by Albers et al. (1999, Mol Microbiol31: 1595–1596) and the thoughts presented in this communication represent interesting alternative views on the export of proteins by archaea.