scholarly journals Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

2010 ◽  
Vol 9 (7) ◽  
pp. 1009-1017 ◽  
Author(s):  
Matthew Cabral ◽  
Christophe Anjard ◽  
Vivek Malhotra ◽  
William F. Loomis ◽  
Adam Kuspa
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Factor ◽  
Late Step ◽  
Unconventional Secretion ◽  
Membrane Bound ◽  
Acyl Coenzyme A ◽  
Mutant Cells

ABSTRACT The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.

scholarly journals Isoprenylcysteine Carboxy Methylation Is Essential for Development in Dictyostelium discoideum

2007 ◽  
Vol 18 (10) ◽  
pp. 4106-4118 ◽  
Author(s):  
Ying Chen ◽  
Kyle J. McQuade ◽  
Xiao-Juan Guan ◽  
Peter A. Thomason ◽  
Michael S. Wert ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Protein Subunit ◽  
Encoding Gene ◽  
Carboxy Terminal ◽  
Mutant Cells ◽  
Do So

Members of the Ras superfamily of small GTPases and the heterotrimeric G protein γ subunit are methylated on their carboxy-terminal cysteine residues by isoprenylcysteine methyltransferase. In Dictyostelium discoideum, small GTPase methylation occurs seconds after stimulation of starving cells by cAMP and returns quickly to basal levels, suggesting an important role in cAMP-dependent signaling. Deleting the isoprenylcysteine methyltransferase-encoding gene causes dramatic defects. Starving mutant cells do not propagate cAMP waves in a sustained manner, and they do not aggregate. Motility is rescued when cells are pulsed with exogenous cAMP, or coplated with wild-type cells, but the rescued cells exhibit altered polarity. cAMP-pulsed methyltransferase-deficient cells that have aggregated fail to differentiate, but mutant cells plated in a wild-type background are able to do so. Localization of and signaling by RasG is altered in the mutant. Localization of the heterotrimeric Gγ protein subunit was normal, but signaling was altered in mutant cells. These data indicate that isoprenylcysteine methylation is required for intercellular signaling and development in Dictyostelium.

scholarly journals Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Mei-Ling Han ◽  
Yan Zhu ◽  
Darren J. Creek ◽  
Yu-Wei Lin ◽  
Dovile Anderson ◽  
...  
Keyword(s):  
Lipid A ◽  
Therapeutic Option ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Metabolomics Data ◽  
Content Type ◽  
In The Wild ◽  
High Level ◽  
Acyl Coenzyme A

ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.

scholarly journals Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

2015 ◽  
Vol 81 (21) ◽  
pp. 7484-7495 ◽  
Author(s):  
Pauline Woan Ying Liew ◽  
Bor Chyan Jong ◽  
Nazalan Najimudin
Keyword(s):  
Cell Surface ◽  
Deletion Mutant ◽  
Azotobacter Vinelandii ◽  
Plant Root ◽  
Hypothetical Protein ◽  
Surface Hydrophobicity ◽  
Root Surface ◽  
Bacterial Cells ◽  
Wild Type ◽  
Content Type

ABSTRACTA proteomic analysis of a soil-dwelling, plant growth-promotingAzotobacter vinelandiistrain showed the presence of a protein encoded by the hypotheticalAvin_16040gene when the bacterial cells were attached to theOryza sativaroot surface. AnAvin_16040deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type.Escherichia colitransformed with the wild-typeAvin_16040gene displayed on its cell surface organized motifs which looked like the S-layer monomers ofA. vinelandii. The recombinantE. colialso demonstrated enhanced adhesion to the root surface.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

2021 ◽  
Vol 17 (8) ◽  
pp. e1009803
Author(s):  
Dipanwita Mitra ◽  
Mohammad H. Hasan ◽  
John T. Bates ◽  
Michael A. Bierdeman ◽  
Dallas R. Ederer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Virus Entry ◽  
Degree Of Polymerization ◽  
Host Cells ◽  
Virus Infectivity ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Host Membrane ◽  
Mutant Cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

scholarly journals Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in Pseudomonas aeruginosa

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Ian T. Hill ◽  
Thomas Tallo ◽  
Matthew J. Dorman ◽  
Simon L. Dove
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Toxic Effects ◽  
Growth Defect ◽  
Wild Type ◽  
Rna Chaperone ◽  
Small Protein ◽  
Content Type ◽  
Transcription Regulator ◽  
Mutant Cells

ABSTRACT Hfq is an RNA chaperone that serves as a master regulator of bacterial physiology. Here we show that in the opportunistic pathogen Pseudomonas aeruginosa, the loss of Hfq can result in a dramatic reduction in growth in a manner that is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using a combination of chromatin immunoprecipitation with high-throughput sequencing and transposon insertion sequencing, we identify the MexT-activated genes responsible for mediating the growth defect of hfq mutant cells. These include a newly identified MexT-controlled gene that we call hilR. We demonstrate that hilR encodes a small protein that is acutely toxic to wild-type cells when produced ectopically. Furthermore, we show that hilR expression is negatively regulated by Hfq, offering a possible explanation for the growth defect of hfq mutant cells. Finally, we present evidence that the expression of MexT-activated genes is dependent upon GshA, an enzyme involved in the synthesis of glutathione. Our findings suggest that Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. Moreover, our results identify glutathione to be a factor important for the in vivo activity of MexT. IMPORTANCE Here we show that the conserved RNA chaperone Hfq is important for the growth of the opportunistic pathogen Pseudomonas aeruginosa. We found that the growth defect of hfq mutant cells is dependent upon the expression of genes that are under the control of the transcription regulator MexT. These include a gene that we refer to as hilR, which we show is negatively regulated by Hfq and encodes a small protein that can be toxic when ectopically produced in wild-type cells. Thus, Hfq can influence the growth of P. aeruginosa by limiting the toxic effects of MexT-regulated genes, including one encoding a previously unrecognized small protein. We also show that MexT activity depends on an enzyme that synthesizes glutathione.

scholarly journals Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

2010 ◽  
Vol 9 (3) ◽  
pp. 387-392 ◽  
Author(s):  
Ewan W. Smith ◽  
Wanessa C. Lima ◽  
Steve J. Charette ◽  
Pierre Cosson
Keyword(s):  
Cell Surface ◽  
Functional Organization ◽  
General Structure ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Rich Medium ◽  
Content Type ◽  
Structure And Dynamics ◽  
Endocytic Compartments ◽  
Mutant Cells

ABSTRACT Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.

scholarly journals A Link between Arabinose Utilization and Oxalotrophy in Bradyrhizobium japonicum

2014 ◽  
Vol 80 (7) ◽  
pp. 2094-2101 ◽  
Author(s):  
Marion Koch ◽  
Nathanaël Delmotte ◽  
Christian H. Ahrens ◽  
Ulrich Omasits ◽  
Kathrin Schneider ◽  
...  
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Host Plants ◽  
Root Nodule ◽  
Coenzyme A ◽  
Root Nodules ◽  
Proteome Analysis ◽  
Wild Type ◽  
Content Type ◽  
Mutant Cells ◽  
Respective Host

ABSTRACTRhizobia have a versatile catabolism that allows them to compete successfully with other microorganisms for nutrients in the soil and in the rhizosphere of their respective host plants. In this study,Bradyrhizobium japonicumUSDA 110 was found to be able to utilize oxalate as the sole carbon source. A proteome analysis of cells grown in minimal medium containing arabinose suggested that oxalate oxidation extends the arabinose degradation branch via glycolaldehyde. A mutant of the key pathway genesoxc(for oxalyl-coenzyme A decarboxylase) andfrc(for formyl-coenzyme A transferase) was constructed and shown to be (i) impaired in growth on arabinose and (ii) unable to grow on oxalate. Oxalate was detected in roots and, at elevated levels, in root nodules of four differentB. japonicumhost plants. Mixed-inoculation experiments with wild-type andoxc-frcmutant cells revealed that oxalotrophy might be a beneficial trait ofB. japonicumat some stage during legume root nodule colonization.

scholarly journals Elimination of defective alpha-factor pheromone receptors.

1997 ◽  
Vol 17 (11) ◽  
pp. 6236-6245 ◽  
Author(s):  
D D Jenness ◽  
Y Li ◽  
C Tipper ◽  
P Spatrick
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Surface ◽  
Half Life ◽  
Structural Defects ◽  
Receptor Protein ◽  
Mutant Form ◽  
Wild Type ◽  
Alpha Factor ◽  
Mutant Cells

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane. When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C.

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