A localized adaptor protein performs distinct functions at the Caulobacter cell poles.
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ABSTRACT:
SUBMITTER: Wang J
PROVIDER: S-EPMC8020655 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Despite their relative simplicity, bacteria have complex anatomy at the subcellular level. At the cell poles of Caulobacter crescentus, a 177-amino acid (aa) protein called PopZ self-assembles into 3D polymeric superstructures. Remarkably, we find that this assemblage interacts directly with at least eight different proteins, which are involved in cell cycle regulation and chromosome segregation. The binding determinants within PopZ include 24 aa at the N terminus, a 32-aa region near the C-terminal homo-oligomeric assembly domain, and portions of an intervening linker region. Together, the N-terminal 133 aa of PopZ are sufficient for interacting with all binding partners, even in the absence of homo-oligomeric assembly. Structural analysis of this region revealed that it is intrinsically disordered, similar to p53 and other hub proteins that organize complex signaling networks in eukaryotic cells. Through live-cell photobleaching, we find rapid binding kinetics between PopZ and its partners, suggesting many pole-localized proteins become concentrated at cell poles through rapid cycles of binding and unbinding within the PopZ scaffold. We conclude that some bacteria, similar to their eukaryotic counterparts, use intrinsically disordered hub proteins for network assembly and subcellular organization.
| S-EPMC5098656 | biostudies-literature
Project description:In Caulobacter crescentus, timely degradation of several proteins by the ClpXP protease is critical for proper cell cycle progression. During the cell cycle, the ClpXP protease, the substrate CtrA, and many other proteins are localized to the stalked pole dependent on a polar interaction hub composed of PopZ protein oligomers. Prior work suggests that the localization of ClpXP, protease substrates, and cofactors is needed for recognition of substrates, such as CtrA, by ClpXP. Here, we formally test this hypothesis by examining the role of PopZ in ClpXP activity and find, surprisingly, that CtrA degradation is enhanced in cells lacking polar localization due to loss of PopZ. The ClpXP adaptor CpdR is required for this enhanced degradation of CtrA and other adaptor-dependent substrates, but adaptor-independent substrate degradation is not affected upon loss of PopZ. We find that overexpression of PopZ also leads to faster degradation of CtrA but is likely due to nonphysiologically relevant recognition of CtrA by ClpXP alone, as loss of CpdR does not affect this enhancement. Our main conclusion is that loss of PopZ, and therefore loss of polar localization, does not result in the loss of ClpXP-regulated proteolysis, as would be predicted from a model which requires polar localization of ClpXP for its activation. Rather, our data point to a model where PopZ normally restrains ClpXP proteolysis by promoting the inactivation of the CpdR adaptor, perhaps through the activity and localization of the CckA kinase.IMPORTANCE Regulated proteolysis is critical for the cell cycle progression of bacteria, such as Caulobacter crescentus According to one model, this regulated proteolysis requires localization of the ClpXP protease at the stalked pole for its subsequent degradation of substrates, such as CtrA. This study offers evidence that supports an alternative model to explain how localization might influence protein degradation. Using a delocalized in vivo system created by the deletion of a polar organizing protein, PopZ, we show that activation of the ClpXP protease is independent of its polar localization. The data point to a role for PopZ in restraining ClpXP activity, likely by controlling the activity of upstream regulators of protease activity, such as CckA, though changes in its localization.
| S-EPMC6153658 | biostudies-literature
Project description:Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.
| S-EPMC2806949 | biostudies-literature
Project description:Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre-divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.
| S-EPMC2908716 | biostudies-literature
Project description:The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket-associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.
| S-EPMC6340215 | biostudies-literature
Project description:The essential process of peptidoglycan synthesis requires two enzymatic activities, transpeptidation and transglycosylation. While the PBP2 and PBP3 transpeptidases perform highly specialized functions that are widely conserved, the specific roles of different glycosyltransferases are poorly understood. For example, Caulobacter crescentus encodes six glycosyltransferase paralogs of largely unknown function. Using genetic analyses, we found that Caulobacter glycosyltransferases are primarily redundant but that PbpX is responsible for most of the essential glycosyltransferase activity. Cells containing PbpX as their sole glycosyltransferase are viable, and the loss of pbpX leads to a general defect in the integrity of the cell wall structure even in the presence of the other five glycosyltransferases. However, neither PbpX nor any of its paralogs is required for the specific processes of cell elongation or division, while the cell wall synthesis required for stalk biogenesis is only partially disrupted in several of the glycosyltransferase mutants. Despite their genetic redundancy, Caulobacter glycosyltransferases exhibit different subcellular localizations. We suggest that these enzymes have specialized roles and normally function in distinct subcomplexes but retain the ability to substitute for one another so as to ensure the robustness of the peptidoglycan synthesis process.
| S-EPMC3807455 | biostudies-literature
Project description:BackgroundBrucella abortus is the etiological agent of a worldwide zoonosis called brucellosis. This alpha-proteobacterium is dividing asymmetrically, and PdhS, an essential histidine kinase, was reported to be an old pole marker.ResultsWe were interested to identify functions that could be recruited to bacterial poles. The Brucella ORFeome, a collection of cloned predicted coding sequences, was placed in fusion with yellow fluorescent protein (YFP) coding sequence and screened for polar localizations in B. abortus. We report that AidB-YFP was systematically localized to the new poles and at constrictions sites in B. abortus, either in culture or inside infected HeLa cells or RAW264.7 macrophages. AidB is an acyl-CoA dehydrogenase (ACAD) homolog, similar to E. coli AidB, an enzyme putatively involved in destroying alkylating agents. Accordingly, a B. abortus aidB mutant is more sensitive than the wild-type strain to the lethality induced by methanesulphonic acid ethyl ester (EMS). The exposure to EMS led to a very low frequency of constriction events, suggesting that cell cycle is blocked during alkylation damage. The localization of AidB-YFP at the new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression of aidB, but not the two other ACAD coding sequences, leads to multiple morphological defects.ConclusionsData reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation.
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Project description:The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory ? subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only ?(1) is involved in ATP synthesis in photosynthesis. The ?(1)-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only ?(2)-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on ?(2)-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in ?(2) is substantially higher than that for ?(1). This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that ?(2)-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that ?(2) originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses.
| S-EPMC3295299 | biostudies-literature
Project description:To colonize surfaces, the bacterium Caulobacter crescentus employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner.
| S-EPMC3926197 | biostudies-literature
Project description:Importin alpha's mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin alpha's, alpha1's, alpha2's, and alpha3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster we describe the isolation and phenotypic characterization of the first animal importin alpha1 mutant. Animal alpha1's are more similar to ancestral plant and fungal alpha1-like genes than to animal alpha2 and alpha3 genes. Male and female importin alpha1 (Dalpha1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dalpha1 mutant phenotypes were rescued by Dalpha1 transgenes, but not by Dalpha2 or Dalpha3 transgenes. Genetic interactions between the ectopic expression of Dalpha1 and the karyopherins CAS and importin beta1 suggest that high nuclear levels of Dalpha1 are deleterious. We conclude that Dalpha1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dalpha1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin alpha-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin alpha-gene family.
| S-EPMC2248332 | biostudies-literature