scholarly journals Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi

Microbiology ◽  
2014 ◽  
Vol 160 (2) ◽  
pp. 316-329 ◽  
Author(s):  
Min Jin Kwon ◽  
Mark Arentshorst ◽  
Markus Fiedler ◽  
Florence L. M. de Groen ◽  
Peter J. Punt ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Reporter Strain ◽  
Expression Platform ◽  
Partial Conservation ◽  
Key Aspects

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.

scholarly journals Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis

2016 ◽  
Vol 18 (9) ◽  
pp. 1268-1284 ◽  
Author(s):  
Joohae Park ◽  
Mark Hulsman ◽  
Mark Arentshorst ◽  
Matthijs Breeman ◽  
Ebru Alazi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Niger ◽  
Genetic Analysis ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis

Molecular genetic analysis of myoC, a Dictyostelium myosin I

1995 ◽  
Vol 108 (3) ◽  
pp. 1093-1103 ◽  
Author(s):  
M.D. Peterson ◽  
K.D. Novak ◽  
M.C. Reedy ◽  
J.I. Ruman ◽  
M.A. Titus
Keyword(s):  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Ultrastructural Organization ◽  
Apparent Lack ◽  
Myosin I ◽  
Family Analysis ◽  
Mutant Cells ◽  
Single Knockout

The protozoan myosin Is are widely expressed actin-based motors, yet their in vivo roles remain poorly understood. Molecular genetic studies have been carried out to determine their in vivo function in the simple eukaryote Dictyostelium, an organism that contains a family of four myosin Is. Here we report the characterization of myoC, a gene that encodes a fifth member of this family. Analysis of the deduced amino acid sequence reveals that the myoC gene encodes a myosin that is homologous to the well-described Acanthamoeba myosin Is as well as to Dictyostelium myoB and -D. The expression pattern of the myoC mRNA is similar to that of myoB and myoD, with a peak of expression at times of maximal cell migration, around 6 hours development. Deletion of the myoB gene has been previously shown to result in mutant cells that are defective in pseudopod extension and phagocytosis. However, no obvious differences in cell growth, development, phagocytosis or motility were detected in cells in which the myoC gene had been disrupted by homologous recombination. F-actin localization and ultrastructural organization also appeared unperturbed in myoC- cells. This apparent ‘lack’ of phenotype in a myosin I single knockout cannot be simply explained by redundancy of function. Our results rather suggest that the present means of assessing myosin I function in vivo are insufficient to identify the unique roles of these actin-based motors.

scholarly journals Shotgun Proteomics of Aspergillus niger Microsomes upon d-Xylose Induction

2010 ◽  
Vol 76 (13) ◽  
pp. 4421-4429 ◽  
Author(s):  
José Miguel P. Ferreira de Oliveira ◽  
Mark W. J. van Passel ◽  
Peter J. Schaap ◽  
Leo H. de Graaff
Keyword(s):  
Mass Spectrometry ◽  
Aspergillus Niger ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Extracellular Enzymes ◽  
Protein Composition ◽  
Shotgun Proteomics ◽  
Small Gtpases ◽  
Hypothetical Proteins ◽  
Extracellular Environment

ABSTRACT Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which—many of them hypothetical proteins—were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

scholarly journals Molecular Genetic Analysis of the Yeast Repressor Rfx1/Crt1 Reveals a Novel Two-Step Regulatory Mechanism

2005 ◽  
Vol 25 (17) ◽  
pp. 7399-7411 ◽  
Author(s):  
Zhengjian Zhang ◽  
Joseph C. Reese
Keyword(s):  
Dna Damage ◽  
Histone Deacetylases ◽  
Molecular Genetic ◽  
Gene Activation ◽  
Molecular Genetic Analysis ◽  
Transcription Activator ◽  
N Terminus ◽  
Inducible Genes ◽  
Repression Domain

ABSTRACT In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were “derepression-defective” mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

scholarly journals Molecular Analysis of Drosophilaeyes absentMutants Reveals Features of the Conserved Eya Domain

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 709-720 ◽  
Author(s):  
Quang T Bui ◽  
John E Zimmerman ◽  
Haixi Liu ◽  
Nancy M Bonini
Keyword(s):  
Protein Sequence ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Special Importance ◽  
Missense Mutations ◽  
Eyes Absent ◽  
Sine Oculis ◽  
Eye Formation

AbstractThe eyes absent (eya) gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other genes of eye development, sine oculis and dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, we have sequenced eya alleles. These mutations have revealed that loss of the entire Eya Domain is null for eya activity, but that alleles with truncations within the Eya Domain display partial function. We then extended the molecular genetic analysis to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminished the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it.

Compound Heterozygosity of the GPIbα Gene Associated with Bernard-Soulier Syndrome

2001 ◽  
Vol 86 (12) ◽  
pp. 1385-1391 ◽  
Author(s):  
Susana Larrucea ◽  
Angela Pastor ◽  
Nora Butta ◽  
Elena Arias-Salgado ◽  
Matilde Ayuso ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Molecular Genetic ◽  
Surface Expression ◽  
Molecular Genetic Analysis ◽  
Paternal Allele ◽  
Platelet Surface ◽  
Cell Lysate ◽  
Translational Frameshift ◽  
Compound Heterozygotes ◽  
Bernard Soulier Syndrome

SummaryWe report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibα and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbα in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbα gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution changing Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbβ and GPIX failed to detect surface expression of the GPIbα mutants. Cells transfected with [1418insT]GPIbα-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast, transfection of [T715A]GPIbα-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbα more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Nonsense Mutation in Exon-19 of GPIIb Associated with Thrombasthenic Phenotype. Failure of GPIIb(Δ597-1008) to Form Stable Complexes with GPIIIa

2002 ◽  
Vol 87 (04) ◽  
pp. 684-691 ◽  
Author(s):  
Elena Arias-Salgado ◽  
Jianming Tao ◽  
Consuelo González-Manchón ◽  
Nora Butta ◽  
Vicente Vicente ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Secretory Pathway ◽  
Stop Codon ◽  
Molecular Genetic ◽  
Surface Expression ◽  
Molecular Genetic Analysis ◽  
Polymorphism Analysis ◽  
Type I ◽  
Single Stranded Conformational Polymorphism ◽  
Exon 19

SummaryWe report the molecular genetic analysis of a patient with thrombasthenic phenotype. The lack of surface platelet GPIIb-IIIa complexes and the presence of GPIIIa suggested it was a case of type I Glanzmann’s thrombasthenia due to a mutation in GPIIb. Single stranded conformational polymorphism analysis (SSCP) of exon-19 of GPIIb showed polymorphic DNA bands. The DNA sequence of exon-19 revealed the presence of a homozygous C1882T transition that changes residue R597 to STOP codon. Since no other mutations were found in either GPIIb or GPIIIa it is concluded that the C1882T substitution in GPIIb is responsible for the thrombasthenic phenotype of the patient. The lack of platelet GPIIb-mRNA in the proband indicates instability of the [C1882T]GPIIb-mRNA. Coexpression of normal GPIIIa and GPIIb(Δ597-1008) in CHO cells failed to show surface expression of GPIIb(Δ597-1008)-IIIa complexes. Immunoprecipitation analysis demonstrated that GPIIb(Δ597-1008) may indeed complex GPIIIa; however, the association is either unstable or incapable of progressing along the secretory pathway.

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