Cloning, Expression, and Purification of Functional Sec11a and Sec11b, Type I Signal Peptidases of the Archaeon
            <i>Haloferax volcanii</i>

Fine, Amir; Irihimovitch, Vered; Dahan, Idit; Konrad, Zvia; Eichler, Jerry

doi:10.1128/jb.188.5.1911-1919.2006

Cited by 34 publications

(31 citation statements)

References 42 publications

(95 reference statements)

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“…Interest in archaeal protein secretion has resulted in studies of various important aspects, including the roles of the Sec and Tat pathways (16,27), the mechanism of action of the various signal peptidases (15,26), numerous attempts to analyze and define archaeal signal peptides (2,5,8), and examination of the archaeal secretome (14,28). Still, the best studied of the archaeal signal peptidases are the type IV prepilin-like peptidases and their role in assembly of archaeal appendages like flagella and pili.…”

Section: Discussionmentioning

confidence: 99%

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

VanDyke

Chaban

et al. 2009

J Bacteriol

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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 ⌬flaK 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 wellcharacterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of Nterminal 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...

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Section: Discussionmentioning

confidence: 99%

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

VanDyke

Chaban

et al. 2009

J Bacteriol

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“…While general aspects of archaeal type I SPase biology have been considered in several recent reviews (Ng et al, 2007; Jarrell et al, 2010; Calo and Eichler, 2011), this enyzme has only been biochemically addressed in a limited number of Archaea to date, including Hfx. volcanii (Fine et al, 2006; Fink-Lavi and Eichler, 2008). In Hfx.…”

Section: Signal Peptide Cleavagementioning

confidence: 99%

“…In Hfx. volcanii , genes encoding two distinct type I SPases, Sec11a ( HVO_2603 ) and Sec11b ( HVO_0002 ) are found (Fine et al, 2006). Of the two, HVO_0002 is an essential gene.…”

Section: Signal Peptide Cleavagementioning

confidence: 99%

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Post-translation modification in Archaea: lessons fromHaloferax volcaniiand other haloarchaea

Eichler

Maupin-Furlow

2013

FEMS Microbiol Rev

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As an ever-growing number of genome sequences appear, it is becoming increasingly clear that factors other than genome sequence impart complexity to the proteome. Of the various sources of proteomic variability, post-translational modifications most greatly serve to expand the variety of proteins found in the cell. Likewise, modulating the rates at which different proteins are degraded also results in a constantly changing cellular protein profile. While both strategies for generating proteomic diversity are adopted by organisms across evolution, the responsible pathways and enzymes in Archaea are often less well described than are their eukaryotic and bacterial counterparts. Studies on halophilic archaea, in particular Haloferax volcanii, originally isolated from the Dead Sea, are helping to fill the void. In this review, recent developments concerning post-translational modifications and protein degradation in the haloarchaea are discussed.

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“…H. volcanii encodes two different SPases I, Sec11a and Sec11b, that can cleave Sec SP in vitro (7). The Sec11b knockout mutant is lethal, whereas the Sec11a knockout mutant is not.…”

Section: Rr/kk (mentioning

confidence: 99%

Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

et al. 2012

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Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase. Subtilisin-like proteases (subtilases) are a functionally diverse superfamily of serine proteases produced by prokaryotes and eukaryotes that include both intra-and extracellular enzymes (30). These proteases serve in a number of biotechnological applications (11,18,24); thus, subtilases from extremophiles represent an attractive resource for both basic and applied science.Secretory subtilases are synthesized as prepro-enzymes consisting of a signal peptide (SP) responsible for secretion, N-terminal and/or C-terminal propeptides, and the catalytic domain (S8 family). Bacterial prepro-subtilisins are processed by removal of the pre-and/or propeptides to render the active extracellular enzymes (30). Comparatively less is known regarding how extracellular proteases are transported through the archaeal membrane and processed into fully active enzymes. In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized (8, 33). Many halophilic bacteria and archaea produce extracellular proteases, however, there is limited information on the pathways used by halophiles to deliver secretory proteases (27,28), and the mechanism that leads to enzyme activation has not been examined.Most extracellular and membrane anchored proteins are expor...

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Cloning, Expression, and Purification of Functional Sec11a and Sec11b, Type I Signal Peptidases of the Archaeon Haloferax volcanii

Cited by 34 publications

References 42 publications

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

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

Post-translation modification in Archaea: lessons fromHaloferax volcaniiand other haloarchaea

Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

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