Antagonistic jacalin-related lectins regulate the size of ER body-type beta-glucosidase complexes in Arabidopsis thaliana

PYK10/BGLU23 is a beta-glucosidase that is a major protein of ER bodies, which are endoplasmic reticulum (ER)-derived organelles that may be involved in defense systems. PYK10 has active and inactive forms. Active PYK10 molecules form large complexes with diameters ranging from 0.65 microm to > 70 microm. We identified three beta-glucosidases (PYK10, BGLU21 and BGLU22), five jacalin-related lectins (JALs) and a GDSL lipase-like protein (GLL) in the purified PYK10 complex. Expression levels of JALs and GLLs were lower in the nai1-1 mutant, which has no ER bodies, than in Col-0. The subcellular localization of PYK10 is predicted to be different from the localizations of JALs and GLLs. This suggests that PYK10 interacts with its partners (JALs and GLLs) when the subcellular structure is destroyed by pathogens. The PYK10 complex was found to be larger in the pbp1-1 and jal22-1 mutants than in Col-0, while it was smaller in the jal23-1, jal31-1 and jal31-2 mutants than in Col-0. These results show that two types of JALs having opposite roles regulate the size of the PYK10 complex antagonistically. We define the two types of lectins as a 'polymerizer-type lectin' and an 'inhibitor-type lectin'. Interestingly, the closest homologs of polymerizer-type lectins (JAL31 and JAL23) were inhibitor-type lectins (PBP1/JAL30 and JAL22). The pairs of polymerizer-type and inhibitor-type lectins reported here are good examples of genes that have evolved new functions after gene duplication (neofunctionalization).     

A novel ER-derived compartment,the ER body,selectively accumulates a beta-glucosidase with an ER-retention signal in Arabidopsis

The ER body is a novel compartment that is derived from endoplasmic reticulum (ER) in Arabidopsis. In contrast to whole seedlings which have a wide distribution of the ER bodies, rosette leaves have no ER bodies. Recently, we reported that wound stress induces the formation of many ER bodies in rosette leaves, suggesting that the ER body plays a role in the defense system of plants. ER bodies were visualized in transgenic plants (GFP-h) expressing green fluorescent protein (GFP) with an ER-retention signal, HDEL. These were concentrated in a 1000-g pellet (P1) of GFP-h plants. We isolated an Arabidopsis mutant, nai1, in which fluorescent ER bodies were hardly detected in whole plants. We found that a 65-kDa protein was specifically accumulated in the P1 fraction of GFP-h plants, but not in the P1 fraction of nai1 plants. N-terminal peptide sequencing revealed that the 65-kDa protein was a beta-glucosidase, PYK10, with an ER-retention signal, KDEL. Immunocytochemistry showed that PYK10 was localized in the ER bodies. Compared with the accumulation of GFP-HDEL, which was associated with both cisternal ER and ER bodies, the accumulation of PYK10 was much more specific to ER bodies. PYK10 was one of the major proteins in cotyledons, hypocotyls and roots of Arabidopsis seedlings, while PYK10 was not detected in rosette leaves that have no ER bodies. These findings indicated that PYK10 is the main component of ER bodies. It is possible that PYK10 produces defense compounds when plants are damaged by insects or wounding.     

The ER body,a novel endoplasmic reticulum-derived structure in Arabidopsis

Plant cells develop various endoplasmic reticulum (ER)-derived structures with specific functions. The ER body, a novel ER-derived compartment in Arabidopsis, is a spindle-shaped structure (approximately 10 microm long and approximately 1 microm wide) that is surrounded by ribosomes. Similar structures were found in many Brassicaceae plants in the 1960s and 1970s, but their main components and biological functions have remained unknown. ER bodies can be visualized in transgenic Arabidopsis expressing the green fluorescent protein with an ER-retention signal. A large number of ER bodies are observed in cotyledons, hypocotyls and roots of seedlings, but very few are observed in rosette leaves. Recently nai1, a mutant that does not develop ER bodies in whole seedlings, was isolated. Analysis of the nai1 mutant reveals that a beta-glucosidase, called PYK10, is the main component of ER bodies. The putative biological function of PYK10 and the inducibility of ER bodies in rosette leaves by wound stress suggest that the ER body functions in the defense against herbivores.     

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active.     

Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.     

The requirement for pneumococcal MreC and MreD is relieved by inactivation of the gene encoding PBP1a

MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae, lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD(Spn)) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae, and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of ΔmreCD mutants, and bypass suppressors accumulate in D39 ΔmreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δpbp1a D39 mutants have smaller diameters than their pbp1a(+) parent or Δpbp2a and Δpbp1b mutants, which lack other class A PBPs and do not show the suppression of ΔmreCD mutations. Suppressed ΔmreCD Δpbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δpbp1a mutants. Quantitative Western blotting showed that MreC(Spn) is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD(Spn) to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD(Spn) direct peripheral PG synthesis and control PBP1a localization or activity.     

Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins.

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding-transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2

Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3beta is a substrate of PYK2 both in vitro and in situ.     

Identification of a genetic element (psr) which negatively controls expression of Enterococcus hirae penicillin-binding protein 5.

Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Rev14) does not produce PBP5, was cloned in pUC18 and sequenced. In the 7.1-kb EcoRI fragment cloned from strain ATCC 9790, an open reading frame (psr) potentially encoding a 19-kDa protein was identified 1 kb upstream of the pbp5 gene. An 87-bp deletion in this element was found in the 7.1-kb EcoRI fragment cloned from strains R40 and Rev14. In addition, several base substitutions were found in the pbp5 genes of strains R40 and Rev14. One of these converted the 42nd codon, TCA, to the stop codon, TAA, in the pbp5 gene of Rev14. Escherichia coli strains were transformed with plasmids carrying the 7.1-kb EcoRI insert or a 2.6-kb HincII insert containing only the pbp5 gene of the three strains. Immunoblotting analysis of proteins expressed by these transformants showed that the 87-bp deletion in psr was associated with the PBP5 overproducer phenotype of strain R40 and the conversion of the TCA codon to the stop codon was associated with the PBP5 nonproducer phenotype of strain Rev14. None of the other nucleotide substitutions had any apparent effect on the level of PBP5 synthesized.     

Endoplasmic reticulum bodies: solving the insoluble

Plant cells produce and accumulate insoluble triglycerides, proteins, and rubber that are assembled into inert, ER-derived organelles broadly termed as ER bodies. ER bodies appear to originate from tubular ER domains that are maintained by cytoskeletal interactions and integral ER proteins. ER bodies sequestering insoluble substances usually are transferred to the vacuole but sometimes remain as cytoplasmic organelles. Some otherwise soluble ER-synthesized proteins are converted to insoluble aggregates to produce ER bodies for transfer to the vacuole. This process constitutes an alternate secretory system to assemble and traffic transport-incompetent insoluble materials.     

Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.     

Localization of p21-activated protein kinase gamma-PAK/Pak2 in the endoplasmic reticulum is required for induction of cytostasis

The intracellular localization and physiological functions of the p21-activated protein kinase gamma-PAK have been examined in human embryonic kidney 293T and COS-7 cells. At 1-4 days post-transfection, cell division is inhibited by the expression of wild type (WT) gamma-PAK and the mutant S490A, whereas cells expressing S490D and the inactive mutants K278R and T402A grow exponentially, indicating a role for gamma-PAK in the induction of cytostasis. WT gamma-PAK and S490A are localized in a region surrounding the nucleus identified as the endoplasmic reticulum (ER), as determined by immunofluorescence, whereas K278R, T402A, and S490D lack localization. As shown by sucrose density gradient centrifugation, WT gamma-PAK, S490A, and endogenous gamma-PAK are distributed among the high density (ER-associated), intermediate density, and low density fractions, whereas the mutants that do not inhibit cell division are present only as soluble enzyme. The amount of endogenous gamma-PAK associated with the particulate fractions is increased 4-fold when cell division is inhibited by ionizing radiation. gamma-PAK in the ER and intermediate density fractions has high specific activity and is active, whereas the soluble form of gamma-PAK has low activity and is activable. The importance of localization of gamma-PAK is supported by data with the C-terminal mutants S490D and Delta 488; these mutants have high levels of protein kinase activity but do not induce cytostasis and are not bound to the ER. A model for the induction of cytostasis by gamma-PAK through targeting of gamma-PAK to the ER is presented in which gamma-PAK activity and Ser-490 are implicated in the regulation of cytostasis.