Conserved residues are critical for <i>Haloferax volcanii</i> archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

Halim, Mohd Farid Abdul; Rodríguez, Ronald; Stoltzfus, Jonathan D.; Duggin, Iain G.; Pohlschröder, Mechthild

doi:10.1111/mmi.13935

Cited by 12 publications

(8 citation statements)

References 37 publications

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“…This opens the way for another hypothesis regarding the ArtA reaction mechanism. In this scenario, ArtA acts similarly to sortase A in bacteria, wherein ArtA cleaves the substrate through thioesterification, forming a thioester acyl᎑enzyme intermediate, which is consistent with the identification of Cys-173 as an active-site residue (13). The nucleophilic attack of an amine resolves this intermediate, but instead of a pentaglycine branched lipid II, the reactive amine nucleophile is archaetidylethanolamine (Fig.…”

Section: Discussionsupporting

confidence: 75%

“…Recently, a novel mechanism was discovered whereby proteins are anchored to the membrane through a lipid moiety covalently attached to a processed C terminus. In archaeal cells, processing and lipid modification of these C-terminal anchored proteins are mediated by enzymes known as archaeosortases, with archaeosortase A (ArtA) of the model archaeon Haloferax volcanii being the most studied example (10)(11)(12)(13). Proteins recognized and processed by H. volcanii ArtA contain a distinct C-terminal tripartite structure consisting of a conserved PGF motif, followed by a hydrophobic domain and then a stretch of positively charged residues.…”

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confidence: 99%

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Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Halim

Schulze

DiLucido

et al. 2020

mBio

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The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

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Section: Discussionsupporting

confidence: 75%

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confidence: 99%

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Halim

Schulze

DiLucido

et al. 2020

mBio

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“…Using this ArtA substrate in combination with site-directed ArtA mutants confirmed the importance of predicted active site residues, which are conserved in ArtA and resemble those of sortases (Abdul Halim et al. 2018 ).…”

Section: Pre-protein Processing and Protein Anchoringmentioning

confidence: 53%

“…Unlike many Euryarchaeota that encode two or three archaeosortase paralogs from different subfamilies, the model archaeon H. volcanii encodes only a single archaeosortase, ArtA (Haft, Payne and Selengut 2012 ; Abdul Halim et al. 2013 , 2018 ). The associated tripartite structure with its PGF motif is present in nine proteins, including the H. volcanii SLG, which was long thought to be anchored to the cell surface via intercalation of a C-terminal TM domain (Lechner and Sumper 1987 ; Sumper et al.…”

Section: Pre-protein Processing and Protein Anchoringmentioning

confidence: 99%

Archaeal cell surface biogenesis

Pohlschröder

Pfeiffer

Schulze

et al. 2018

FEMS Microbiology Reviews

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Cell surfaces are critical for diverse functions across all domains of life, from cell-cell communication and nutrient uptake to cell stability and surface attachment. While certain aspects of the mechanisms supporting the biosynthesis of the archaeal cell surface are unique, likely due to important differences in cell surface compositions between domains, others are shared with bacteria or eukaryotes or both. Based on recent studies completed on a phylogenetically diverse array of archaea, from a wide variety of habitats, here we discuss advances in the characterization of mechanisms underpinning archaeal cell surface biogenesis. These include those facilitating co- and post-translational protein targeting to the cell surface, transport into and across the archaeal lipid membrane, and protein anchoring strategies. We also discuss, in some detail, the assembly of specific cell surface structures, such as the archaeal S-layer and the type IV pili. We will highlight the importance of post-translational protein modifications, such as lipid attachment and glycosylation, in the biosynthesis as well as the regulation of the functions of these cell surface structures and present the differences and similarities in the biogenesis of type IV pili across prokaryotic domains.

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“…Its genome has been sequenced and carefully annotated (Hartman et al, 2010, Pfeiffer et al, 2008a, Pfeiffer and Oesterhelt, 2015. A plethora of biological aspects have been successfully tackled in this species, with examples including DNA replication (Perez-Arnaiz et al, 2020); cell division and cell shape (Turkowyd et al, 2020, Walsh et al, 2019, de Silva et al, 2021, Duggin et al, 2015, Liao et al, 2021; metabolism (Brasen and Schonheit, 2001, Johnsen et al, 2009, Pickl et al, 2012, Sutter et al, 2016, Reinhardt et al, 2019, Kuprat et al, 2021, Kuprat et al, 2020, Sutter et al, 2020, Tästensen et al, 2020; protein secretion (Abdul-Halim et al, 2020, Abdul Halim et al, 2018, Abdul Halim et al, 2013, Storf et al, 2010; motility and biofilms (Schiller et al, 2020, Pohlschroder and Esquivel, 2015, Li et al, 2020, Collins et al, 2020, Quax et al, 2018, Nussbaum et al, 2020; mating (Shalev et al, 2017); signalling (Braun et al, 2019); virus defence (Maier et al, 2019); proteolysis (Reuter and Maupin-Furlow, 2004, Reuter et al, 2010, Prunetti et al, 2014, Cerletti et al, 2014, Costa et al, 2018; posttranslati...…”

Section: Introductionmentioning

confidence: 99%

Open issues for protein function assignment inHaloferax volcaniiand other halophilic archaea

Pfeiffer

Dyall‐Smith

2021

Preprint

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Background: Annotation ambiguities and annotation errors are a general challenge in genomics. While a reliable protein function assignment can be obtained by experimental characterization, this is expensive and time-consuming, and the number of such Gold Standard Proteins (GSP) with experimental support remains very low compared to proteins annotated by sequence homology, usually through automated pipelines. Even a GSP may give a misleading assignment when used as a reference: the homolog may be close enough to support isofunctionality, but the substrate of the GSP is absent from the species being annotated. In such cases the enzymes cannot be isofunctional. Here, we examine a variety of such issues in halophilic archaea (class Halobacteria), with a strong focus on the model haloarchaeon Haloferax volcanii. Results: Annotated proteins of Hfx. volcanii were identified for which public databases tend to assign a function that is probably incorrect. In some cases, an alternative, probably correct, function can be predicted or inferred from the available evidence but this has not been adopted by public databases because experimental validation is lacking. In other cases, a probably invalid specific function is predicted by homology, and while there is evidence that this assigned function is unlikely, the true function remains elusive. We list 50 of those cases, each with detailed background information so that a conclusion about the most likely biological function can be drawn. For reasons of brevity and comprehension, only key aspects are listed in the main text, with detailed information being provided in a corresponding section of the Supplementary Material. Conclusions: Compiling, describing and summarizing these open annotation issues and functional predictions will benefit the scientific community in the general effort to improve the evaluation of protein function assignments and more thoroughly detail them. By highlighting the gaps and likely annotation errors currently in the databases, we hope this study will provide a framework for experimentalists to sytematically confirm (or disprove) our function predictions or to uncover yet unexpected functions.

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Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

Cited by 12 publications

References 37 publications

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Archaeal cell surface biogenesis

Open issues for protein function assignment inHaloferax volcaniiand other halophilic archaea

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