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Query: UNIPROT:P06889 (Mol)
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It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 alone or by 8 M urea, 6 M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non-sedimentable PulG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.
Mol Microbiol 1993 Nov
PMID:The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex. 796 43

pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide-binding protein. Subcellular fractionation indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non-hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucleotide-binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate-rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide-binding proteins. Altogether, these results indicate that PulE might belong to a new family of nucleotide-binding proteins. The protein could not be cross-linked to the photoactivatable ATP analogue azido-ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild-type PulE protein. Evidence presented suggests that PulE might be a homodimer.
Mol Microbiol 1994 Apr
PMID:Molecular characterization of PulE, a protein required for pullulanase secretion. 805 53

High glycogen content and abnormal mitochondria have been seen in muscles from RN- carrier pigs in a previous work. Glycogen synthase, branching enzyme, phosphorylase and debranching enzyme activities, and mitochondrial characteristics were studied in normal and RN- carrier pigs. Branching enzyme activity was higher (P < 0.01) and glycogen synthase activity tended to be higher in longissimus dorsi muscle from RN- carrier pigs compared to normal pigs. There were no differences in the activities of either phosphorylase and debranching enzyme between both types of pigs. Citrate synthase activity and mitochondrial respiration were slightly higher in muscle from RN- pigs compared to normal pigs. Glycogen content in muscle from RN- pigs could result from the imbalance between anabolic and catabolic enzyme activities of glycogen metabolism. The higher specific activity in mitochondria of RN- pigs muscle might be the compensatory effect of an abnormal glycolytic metabolism.
Comp Biochem Physiol Biochem Mol Biol 1994 Jul
PMID:Enzyme activities of glycogen metabolism and mitochondrial characteristics in muscles of RN- carrier pigs (Sus scrofa domesticus). 808 56

The signal sequence of the Klebsiella oxytoca puIG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the puIO gene product processes prePuIG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PuIG signal sequence. Like most type IV pilins, processed PuIG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePuIG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePuIG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePuIG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePuIG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.
Mol Microbiol 1993 Jul
PMID:Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca. 841 82

Two genes from Thermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (Pg1A). The xynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The gene pglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and Pg1A are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of Pg1A and AmyB from T. thermosulfurigenes EM1.
Mol Gen Genet 1996 Sep 25
PMID:Characterization of genes from Thermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains. 887 52

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.
Mol Microbiol 1996 Dec
PMID:The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions. 897 17

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.
Mol Cell Biol 1997 Feb
PMID:Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation. 900 Dec 35

In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIV(f1) specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIV(IKe). In native pIV(IKe), two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIV(f1) and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.
J Mol Biol 1997 Mar 14
PMID:Module swaps between related translocator proteins pIV(f1), pIV(IKe) and PulD: identification of a specificity domain. 908 75

The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to < or = 10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.
Mol Microbiol 1997 May
PMID:Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway. 917 40

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.
Mol Microbiol 1997 May
PMID:The C-terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function. 917 41


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