scholarly journals Knocking Out Bcsas1 in Botrytis cinerea Impacts Growth, Development, and Secretion of Extracellular Proteins, Which Decreases Virulence

2014 ◽  
Vol 27 (6) ◽  
pp. 590-600 ◽  
Author(s):  
Zhanquan Zhang ◽  
Guozheng Qin ◽  
Boqiang Li ◽  
Shiping Tian
Keyword(s):  
Botrytis Cinerea ◽  
Secretory Pathway ◽  
Pathogenic Fungi ◽  
Extracellular Proteins ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Extracellular Medium ◽  
Expression Of Genes ◽  
Transport Vesicles ◽  
Genes Encoding

Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

scholarly journals An Experimental Approach for the Identification of Conserved Secreted Proteins in Trypanosomatids

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Rosa M. Corrales ◽  
Françoise Mathieu-Daudé ◽  
Déborah Garcia ◽  
Simone F. Brenière ◽  
Denis Sereno
Keyword(s):  
Secretory Pathway ◽  
Experimental System ◽  
Bioinformatic Analysis ◽  
Extracellular Proteins ◽  
Peptide Sequence ◽  
Secreted Proteins ◽  
Extracellular Medium ◽  
Signal Peptide Sequence ◽  
A Genome ◽  
Genes Encoding

Extracellular factors produced byLeishmania spp., Trypanosoma cruzi,andTrypanosoma bruceiare important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of theT. cruzigenome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence inL. infantum, L. major,andT. bruceiwere transfected intoL. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for twoL. infantumorthologs proteins using the same experimental system. Infectivity studies of transgenicLeishmaniaparasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2022 ◽  
Author(s):  
Javier Manzano-Lopez†* ◽  
Sofia Rodriguez-Gallardo† ◽  
Susana Sabido-Bozo† ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2021 ◽  
Author(s):  
Javier Manzano-Lopez † ◽  
Sofia Rodriguez-Gallardo † ◽  
Susana Sabido-Bozo ◽  
Ana Maria Perez-Linero ◽  
Rafael Lucena ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

scholarly journals Epstein-Barr Virus Exploits the Secretory Pathway to Release Virions

2020 ◽  
Vol 8 (5) ◽  
pp. 729
Author(s):  
Asuka Nanbo
Keyword(s):  
Epstein Barr Virus ◽  
Secretory Pathway ◽  
Lytic Cycle ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Extracellular Milieu ◽  
Barr Virus ◽  
Intracellular Compartments ◽  
Epstein Barr

Herpesvirus egress mechanisms are strongly associated with intracellular compartment remodeling processes. Previously, we and other groups have described that intracellular compartments derived from the Golgi apparatus are the maturation sites of Epstein-Barr virus (EBV) virions. However, the mechanism by which these virions are released from the host cell to the extracellular milieu is poorly understood. Here, I adapted two independent induction systems of the EBV lytic cycle in vitro, in the context of Rab GTPase silencing, to characterize the EBV release pathway. Immunofluorescence staining revealed that p350/220, the major EBV glycoprotein, partially co-localized with three Rab GTPases: Rab8a, Rab10, and Rab11a. Furthermore, the knockdown of these Rab GTPases promoted the intracellular accumulation of viral structural proteins by inhibiting its distribution to the plasma membrane. Finally, the knockdown of the Rab8a, Rab10, and Rab11a proteins suppressed the release of EBV infectious virions. Taken together, these findings support the hypothesis that mature EBV virions are released from infected cells to the extracellular milieu via the secretory pathway, as well as providing new insights into the EBV life cycle.

scholarly journals The route of secretion of procollagen. The influence of αα′-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells

1976 ◽  
Vol 156 (1) ◽  
pp. 81-90 ◽  
Author(s):  
R Harwood ◽  
M E Grant ◽  
D S Jackson
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Smooth Endoplasmic Reticulum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extracellular Proteins ◽  
Secretory Process ◽  
Antimycin A ◽  
Embryonic Chick ◽  
Extracellular Medium ◽  
Cartilage Cells

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha′-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.

scholarly journals Yos1p Is a Novel Subunit of the Yip1p–Yif1p Complex and Is Required for Transport between the Endoplasmic Reticulum and the Golgi Complex

2005 ◽  
Vol 16 (4) ◽  
pp. 1673-1683 ◽  
Author(s):  
Matthew Heidtman ◽  
Catherine Z. Chen ◽  
Ruth N. Collins ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Rab Gtpases ◽  
Reading Frame ◽  
Multicopy Suppressor ◽  
Transport Vesicles ◽  
Identification And Characterization

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p–Yif1p complex.

scholarly journals The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity

2010 ◽  
Vol 191 (3) ◽  
pp. 479-492 ◽  
Author(s):  
Rebecca M. Fox ◽  
Caitlin D. Hanlon ◽  
Deborah J. Andrew
Keyword(s):  
Transcription Factors ◽  
Secretory Pathway ◽  
Cell Types ◽  
Secretory Cells ◽  
Cell Type ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Cell Type Specific ◽  
General Protein ◽  
Secretory Capacity

Secretion occurs in all cells, with relatively low levels in most cells and extremely high levels in specialized secretory cells, such as those of the pancreas, salivary, and mammary glands. How secretory capacity is selectively up-regulated in specialized secretory cells is unknown. Here, we find that the CrebA/Creb3-like family of bZip transcription factors functions to up-regulate expression of both the general protein machinery required in all cells for secretion and of cell type–specific secreted proteins. Drosophila CrebA directly binds the enhancers of secretory pathway genes and is both necessary and sufficient to activate expression of every secretory pathway component gene examined thus far. Microarray profiling reveals that CrebA also up-regulates expression of genes encoding cell type–specific secreted components. Finally, we found that the human CrebA orthologues, Creb3L1 and Creb3L2, have the ability to up-regulate the secretory pathway in nonsecretory cell types.

scholarly journals Actin Is Required for Cellular Development and Virulence of Botrytis cinerea via the Mediation of Secretory Proteins

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Hua Li ◽  
Zhanquan Zhang ◽  
Guozheng Qin ◽  
Chang He ◽  
Boqiang Li ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Virulence Factors ◽  
Proteomic Analysis ◽  
Crucial Role ◽  
Pathogenic Fungi ◽  
Extracellular Proteins ◽  
Secretory Proteins ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Vital Component

ABSTRACT Actin is a vital component of the cytoskeleton of living cells and is involved in several complex processes. However, its functions in plant-pathogenic fungi are largely unknown. In this paper, we found that deletion of the Botrytis cinerea actin gene bcactA reduced growth and sporulation of B. cinerea and lowered virulence. Based on iTRAQ (isobaric tags for relative and absolute quantification)-based proteomic analysis, we compared changes of the secretome in ΔbcactA and wild-type strains. A total of 40 proteins exhibited significant differences in abundance in ΔbcactA mutants compared with the wild type. These proteins included 11 down-accumulated cell wall-degrading enzymes (CWDEs). Among them, two CWDEs, cellobiohydrolase (BcCBH) and β-endoglucanase (BcEG), were found to contribute to the virulence of B. cinerea, indicating that bcactA plays a crucial role in regulating the secretion of extracellular virulence factors. These findings unveil previously unknown functions of BcactA to mediate the virulence of B. cinerea and provide new mechanistic insights into the role of BcactA in the complex pathogenesis of B. cinerea. IMPORTANCE The cytoskeleton is an important network that exists in cells of all domains of life. In eukaryotic cells, actin is a vital component of the cytoskeleton. Here, we report that BcactA, an actin protein in B. cinerea, can affect the growth, sporulation, and virulence of B. cinerea. Furthermore, iTRAQ-based proteomic analysis showed that BcactA affects the abundance of 40 extracellular proteins, including 11 down-accumulated CWDEs. Among them, two CWDEs, cellobiohydrolase (BcCBH) and β-endoglucanase (BcEG), contributed to the virulence of B. cinerea, indicating that bcactA plays a crucial role in regulating extracellular virulence factors. These findings unveil previously unknown functions of BcactA in mediating growth, sporulation, and virulence of B. cinerea.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2021 ◽  
Author(s):  
Javier Manzano-Lopez † ◽  
Sofia Rodriguez-Gallardo † ◽  
Susana Sabido-Bozo ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

Sign in / Sign up

Export Citation Format

Share Document