Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.The bacterial type IV prepilin peptidase (TFPP) is a well-characterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of N-terminal signal peptides from prepilins and pseudopilins, prior to their incorporation into the type IV pilus structure (22, 30, 31). The prepilin peptidase is also responsible for the processing of prepilin-like proteins needed for type II secretion (22). In Archaea, the existence of bacterial TFPP-like enzymes has also been reported, and they have been most extensively studied in relation to the assembly of the archaeal flagellum. In the euryarchaeotes Methanococcus maripaludis and Methanococcus voltae, the preflagellin peptidase FlaK was demonstrated to be responsible for cleaving the N-terminal signal peptide from the preflagellin prior to its incorporation into the growing flagellar filament, a step essential to flagellar assembly (6, 7, 26). In Sulfolobus solfataricus, an acidophilic crenarchaeote, the equivalent enzyme, PibD, was also shown to process preflagellins (4). Site-directed mutagenesis of FlaK and PibD demonstrated that both aspartic acid residues that aligned with aspartic acid residues essential for bacterial TFPP activity were also essential in the archaeal enzymes (6, 32), indicating that the two archaeal peptidases belong with the bacterial TFPPs in this novel family of aspartic acid proteases (20). More recently, an additional archaeal TFPP was found to be required for cleavage of the prepilin substrates (33) that are assembled into the unique pili of M. maripaludis (37).The substrate specificity of the archaeal preflagellin peptidase remains an open question. Like prepilin peptidases, FlaK in M. voltae has stringent requirements for the amino acids surrounding the cleavage site of the substrate, especially the −1 glycine, −2 and −3 lysines, and the +3 glycine (numbers given relative to the cleavage site) (35); the last position was conserved in all archaeal flagellins (25). Upon N-terminal sequence alignment of all available archaeal flagellin amino acid sequences at the predicted cleavage site, it was found that most archaeal preflagellin signal peptides are quite conserved in length, with the typical flagellin signal peptide being 11 to 12 amino acids in length (Table ​(Table1).1). It is speculated that while a certain amount of flexibility might exist, some optimum and minimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane (18). A recent study examining possible type IV pilin-like substrates in archaea using the FlaFind program indicated that such substrates may be more widespread than initially thought (33). Since in Methanococcus the pilins are processed by a second TFPP (EppA) (33), it is very possible that the preflagellins might be the only substrates of FlaK in these archaea.

TABLE 1.

N-terminal amino acid alignment of selected archaeal flagellin sequences^a

Organism	Flagellin	N-terminal sequence
Archaeoglobus fulgidus	FlaB1	MGMRFLKNEKGFTGLEAAIVLIAFVTVAAVFSYVLL
Aeropyrum pernix	FlaB1	MRRRRGIVGIEAAIVLIAFVIVAAALAFVAL
Haloarcula marismortui	FlaA	MFEKIANENERGQVGIGTLIVFIAMVLVAAIAAGVLI
Halobacterium salinarum	FlgA1	MFEFITDEDERGQVGIGTLIVFIAMVLVAAIAAGVLI
Methanocaldococcus jannaschii	FlaB1	MKVFEFLKGKRGAMGIGTLIIFIAMVLVAAVAAAVLI
Methanococcoides burtonii	Fla	MKANKHLMMNNDRAQAGIGTLIIFIAMVLVAAVAAAVLI
Methanococcus aeolicus	Fla	MNLEHFSFLKNKKGAMGIGTLIIFIAMVLVAAVAASVLI
Methanococcus maripaludis	FlaB1	MKIKEFLKTKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanococcus vannielii	FlaB1	MSVKNFMNNKKGDSGIGTLIVFIAMVLVAAVAASVLI
Methanococcus voltae	FlaB2	MKIKEFMSNKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanothermococcus thermolithotrophicus	FlaB1	MKIAQFIKDKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanogenium marisnigri	Fla	MKRQFNDNAFTGLEAAIVLIAFIVVAAVFSYVVL
Methanospirillum hungatei	Fla	MNNEDGFSGLEAMIVLIAFVVVAAVFAYATL
Natrialba magadii	FlaB1	MFEQNDDRDRGQVGIGTLIVFIAMVLVAAIAAGVLI
Natronomonas pharaonis	Flg1	MFETLTETKERGQVGIGTLIVFIALVLVAAIAAGVLI
Pyrococcus abyssi	FlaB1	MRRGAIGIGTLIVFIAMVLVAAVAAGVLI
Pyrococcus furiosus	Fla	MKKGAIGIGTLIVFIAMVLVAAVAAGVLI
Pyrococcus horikoshii	FlaB1	MRRGAIGIGTLIVFIAMVLVAAVAAAVLI
Sulfolobus solfataricus	Fla	MNSKKMLKEYNKKVKRKGLAGLDTAIILIAFIITASVLAYVAI
Sulfolobus tokodaii	Fla	MGAKNAIKKYNKIVKRKGLAGLDTAIILIAFIITASVLAYVAI
Thermococcus kodakarensis	FlaB1	MKTRTRKGAVGIGTLIVFIAMVLVAAVAAAVLI
Thermoplasma acidophilum	Fla	MRKVFSLKADNKAETGIGTLIVFIAMVLVAAVAATVLI
Thermoplasma volcanium	Fla	MYIVKKMPILKLLNSIKRIFKTDDSKAESGIGVLIVFIAMILVAAVAASVLI

Open in a separate window^aIn all organisms listed, except Sulfolobus, there are multiple flagellins but only a single example is shown. The signal peptide is shown in boldface type. In some cases, analyses of the amino acid sequences of the signal peptides with unusual lengths revealed in-frame methionines or alternative start sites (underlined) that, if they represent the true translation start site, would result in signal peptides of more typical lengths. For S. solfataricus, Albers et al. (4) used the internal start site to give a signal peptide of 13 amino acids and demonstrated signal peptide processing.Studies on PibD in S. solfataricus, however, present interesting disparities. A recent genomic survey revealed a surprisingly large group of proteins possessing type IV pilin-like signal peptides in Sulfolobus compared to other archaea (2, 33). Besides the preflagellins, other substrates for PibD include pilins and proteins involved in sugar binding. Deletions of pibD appear to be nonviable (1), unlike the case for flaK, reinforcing the role of pibD in processes other than flagellum and pilus formation. Site-directed mutagenesis on the glucose-binding protein precursor (GlcS) signal peptide revealed that a wide variety of substitutions around the cleavage site still permitted processing. The allowed substitutions were consistent with the signal peptide sequences of a list of proposed PibD substrates, some of which have predicted signal peptides as short as 3 amino acids (4). Based on the observation that homologues of S. solfataricus sugar-binding proteins that contain type IV prepilin-like sequences were absent in the genome of another species of Sulfolobus, Sulfolobus tokodaii, it was speculated that S. solfataricus PibD may have undergone a specialization allowing for a broader substrate specificity (4). However, whether the extremely short signal peptides would be functional and recognizable as preflagellin peptidase substrates remains to be biochemically demonstrated.Although the typical flagellin signal peptide is 11 to 12 amino acids in length, a small number of archaeal preflagellins contain signal peptides of unusual lengths. Some are annotated to be unusually long (e.g., MJ0893 of Methanocaldococcus jannaschii and Ta1407 of Thermoplasma acidophilum) (Table ​(Table1).1). These sequences, however, contain in-frame alternative translational start sites that, if they correspond to true translation start sites, would result in signal peptides more typical in length. On the other hand, organisms with preflagellins predicted to possess unusually short signal peptides of 4 to 6 amino acids include Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, and Aeropyrum pernix (Table ​(Table1).1). These unusual signal peptides are deduced exclusively from gene sequences. Biochemical or genetic data to explain these peculiarities are still lacking. Assuming that the annotations of these genes are accurate, this would suggest that certain archaeal TFPP-like enzymes possess the capacity to process these much shorter signal peptides.In this study, for the first time, a systematic evaluation of critical signal peptide length for recognition and cleavage by two very different archaeal TFPP-like signal peptidases, M. voltae FlaK and S. solfataricus PibD, is reported.