Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens

Abstract

Methanococcus voltae is a mesophilic archaeon with flagella composed of flagellins that are initially made with 11-or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits. Escherichia coli membranes containing the expressed M. voltae preflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species. M. voltae preflagellin peptidase activity was optimal at 37°C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.All of the major subgroupings of archaea, including methanogens, extreme halophiles, and sulfur-dependent thermophiles and hyperthermophiles, have members that possess flagella that look superficially like bacterial flagella (10). However, recent evidence has indicated that the archaeal flagellum is a unique motility structure, distinct from that of bacteria in composition and likely assembly (4, 10). One major distinction is that the assembly of archaeal flagella requires the posttranslational cleavage of a short (11-or 12-amino-acid) leader peptide (2, 13) from the precursor form of the flagellin monomer (preflagellin) before its incorporation into the growing filament. Bacterial flagellins are not made with leader peptides (12).Previously, in Methanococcus voltae, four flagellin genes (flaA, flaB1, flaB2, and flaB3) carried by two transcriptional units were identified (13). One transcriptional unit contains only flaA; the other, polycistronic transcriptional unit (at least 5.4 kb in length), containing flaB1, flaB2, flaB3, and a number of presumed flagellum accessory genes, initiates at flaB1 and extends to at least the end of flaG (13; D. P. Bayley, J. D. Correia, and K. F. Jarrell, unpublished data). N-terminal (14), transcriptional (13), and mutational (9) analyses have provided evidence that flaB1 and flaB2 encode the major flagellins in M. voltae. Comparison of the N-terminal sequence of one purified flagellin with the amino acid sequence predicted from the gene sequence (13) confirmed the presence of a 12-amino-acid leader peptide. Similarly, in the related methanogen Methanococcus vannielii, comparison of the N-terminal sequences obtained from the two major flagellins of purified flagellar filaments with the deduced amino acid sequence of the cloned genes indicated the presence of 12-amino-acid leader peptides on the FlaB1 and FlaB2 flagellins (2). The presence of leader peptides on archaeal flagellins indicated that enzymatic activity must be present in archaeal cells to process the preflagellins.Two different substrates were used for the determination of preflagellin peptidase activity. One preflagellin substrate was prepared by the expression of M. voltae Fla...

Posttranslational Protein Modification inArchaea

“…The unique signal sequences of archaeal flagellins are removed by the actions of a signal peptidase reminiscent of bacterial type IV prepilin peptidases (17,75), exemplified by Pseudomonas aeruginosa PilD (419). Those translocated nonflagellar Sulfolobus solfataricus proteins bearing the archaeal flagellin signal sequence also rely on an archaeal version of the bacterial type IV prepilin peptidase, termed PibD (peptidase involved in biogenesis of prepilin-like proteins), for their processing (2, 6).…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…FlaK and PibD have conserved C-terminal Asp residues, suggesting that the archaeal peptidases may likewise function as aspartyl proteases. FlaK peptidase activity was detected in M. voltae membrane fractions (109), and site-directed mutagenesis of the conserved C-terminal Asp residues abrogated peptidase activity (39). The recently solved FlaK crystal structure is proposed to show an inactive conformation, because the two Asp residues are relatively far apart (188).…”

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

“…To remove this, E. coli membranes containing FlaB2 were used as substrate in an in vitro preflagellin peptidase assay using M. voltae membranes as a source of the preflagellin peptidase. Both the unprocessed and processed forms of FlaB2 are detected in a Western blot using anti-flagellin antisera (17).…”

“…Preflagellin Peptidase Reaction-A non-glycosylated form of FlaB2 was produced in Escherichia coli as described previously (17). This form has the signal peptide of FlaB2 still attached.…”