ABSTRACT 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 Δ artA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the Δ hvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the Δ hvpssA and Δ hvpssD 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.
more »
« less
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
Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in
- NSF-PAR ID:
- 10055063
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Microbiology
- Volume:
- 108
- Issue:
- 3
- ISSN:
- 0950-382X
- Page Range / eLocation ID:
- p. 276-287
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of
S ‐acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram‐negative pathogenFrancisella tularensis , exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding β‐tongue and substrate‐binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/β hydrolase structure and a potential allosteric binding site within human APTs. -
Ganesan, A (Ed.)more » « less
Mammalian protein arginine methyltransferase 7 (PRMT7) has been shown to target substrates with motifs containing two arginine residues separated by one other residue (RXR motifs). In particular, the repression domain of human histone H2B (29-RKRSR-33) has been a key substrate in determining PRMT7 activity. We show that incubating human PRMT7 and [3H]-AdoMet with full-length
Xenopus laevis histone H2B, containing the substitutions K30R and R31K (RKRSR to RRKSR), results in greatly reduced methylation activity. Using synthetic peptides, we have now focused on the enzymology behind this specificity. We show for the human and Xenopus peptide sequences 23–37 the difference in activity results from changes in the Vmaxrather than the apparent binding affinity of the enzyme for the substrates. We then characterized six additional peptides containing a single arginine or a pair of arginine residues flanked by glycine and lysine residues. We have corroborated previous findings that peptides with an RXR motif have much higher activity than peptides that contain only one Arg residue. We show that these peptides have similar apparent kmvalues but significant differences in their Vmaxvalues. Finally, we have examined the effect of ionic strength on these peptides. We found the inclusion of salt had little effect on the Vmaxvalue but a considerable increase in the apparent kmvalue, suggesting that the inhibitory effect of ionic strength on PRMT7 activity occurs largely by decreasing apparent substrate-enzyme binding affinity. In summary, we find that even subtle substitutions in the RXR recognition motif can dramatically affect PRMT7 catalysis. -
more » « less
Summary A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone‐protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix.
Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N‐terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)).
Strong synergistic interactions were observed between the CLP protease system (
clpr1‐2 ,clpr2‐1 ,clpc1‐1 ,clpt1 ,clpt2) and both PREP homologs (prep1 ,prep2 ) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N‐terminal proteomess howed that many nuclear encoded chloroplast proteins have alternatively processed N‐termini inprep1prep2 ,clpt1clpt2 andprep1prep2clpt1clpt2 .Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.
-
Phosphorylation of Inhibitor of κB (IκB) proteins by IκB Kinase β (IKKβ) leads to IκB degradation and subsequent activation of nuclear factor κB transcription factors. Of particular interest is the IKKβ-catalyzed phosphorylation of IκBα residues Ser32 and Ser36 within a conserved destruction box motif. To investigate the catalytic mechanism of IKKβ,we performed pre–steady-state kinetic analysis of the phosphorylation of IκBα protein substrates catalyzed by constitutively active, human IKKβ. Phosphorylation of full-length IκBα catalyzed by IKKβ was characterized by a fast exponential phase followed by a slower linear phase. The maximum observed rate (kp) of IKKβ-catalyzed phosphorylation of IκBα was 0.32 s−1 and the binding affinity of ATP for the IKKβ IκBα complex (Kd) was 12 μM. Substitution of either Ser32 or Ser36 with Ala, Asp, or Cys reduced the amplitude of the exponential phase by approximately 2-fold. Thus, the exponential phase was attributed to phosphorylation of IκBα at Ser32 and Ser36, whereas the slower linear phase was attributed to phosphorylation of other residues. Interestingly, the exponential rate of phosphorylation of the IκBα(S32D) phosphomimetic amino acid substitution mutant was nearly twice that of WT IκBα and 4-fold faster than any of the other IκBα amino acid substitution mutants, suggesting that phosphorylation of Ser32 increases the phosphorylation rate of Ser36. These conclusions were supported by parallel experiments using GST-IκBα(1–54) fusion protein substrates bearing the first 54 residues of IκBα. Our data suggest a model wherein, IKKβ phosphorylates IκBα at Ser32 followed by Ser36 within a single binding event.more » « less
