scholarly journals CRISPR-based bioengineering of the Transferrin Receptor revealed a role for Rab7 in the biosynthetic secretory pathway

2020 ◽  
Author(s):  
Maika S. Deffieu ◽  
Ieva Cesonyte ◽  
François Delalande ◽  
Gaelle Boncompain ◽  
Cristina Dorobantu ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Quantitative Proteomics ◽  
Secretory Pathway ◽  
Gene Editing ◽  
The Other ◽  
Rab Gtpase ◽  
Secretory Vesicles ◽  
Proteomics Data ◽  
Transmembrane Receptors ◽  
Intermediate Compartment

AbstractThe regulated secretory trafficking of neosynthesized transmembrane receptors is particularly challenging to investigate as it is under-represented at steady state compared to the abundance of the other trafficking routes. Here, we combined the retention using selective hook (RUSH) system to a CRISPR/Cas9 gene editing approach (eRUSH) to identify molecular players involved in the trafficking of neosynthesized Transferrin Receptor (TfR) en route to the plasma membrane (PM). TfR-eRUSH monoclonal cells expressing endogenous, ER-retainable and fluorescent TfR were engineered and characterized. Spatiotemporal quantitative proteomics of TfR-eRUSH cells allowed the identification of molecular partners associated with TfR-containing membranes and provided a comprehensive list of potential regulators, co-trafficking cargos, and enriched pathways. Furthermore, we chose to focus our attention on the Rab GTPase family members for their function as vesicle trafficking regulators and performed a Rab-targeted siRNA screen that we correlated to our proteomics data. Unexpectedly, we identified Rab7-harboring vesicles as an intermediate compartment of the Golgi-to-PM transport of the neosynthetic TfR. These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles, also involved in Golgi-to-PM transport. However, Rab6A-TfR vesicles delivered TfR directly to the PM, while in contrast, Rab7A was transiently associated to neosynthetic TfR-containing post-Golgi vesicles but dissociated before PM vesicle fusion. Together, our study proposes the eRUSH as a powerful tool to further study the secretory pathway and reveals an unforeseen role for Rab7 in the neosynthetic transport of the TfR, highlighting the diversity of the secretory vesicles’ nature for a given cargo.

scholarly journals Rab7-harboring vesicles are carriers of the transferrin receptor through the biosynthetic secretory pathway

2021 ◽  
Vol 7 (2) ◽  
pp. eaba7803
Author(s):  
Maika S. Deffieu ◽  
Ieva Cesonyte ◽  
François Delalande ◽  
Gaelle Boncompain ◽  
Cristina Dorobantu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Transport ◽  
Transferrin Receptor ◽  
Intracellular Trafficking ◽  
Secretory Pathway ◽  
Gene Editing ◽  
The Other ◽  
Secretory Vesicles ◽  
Important Intermediate ◽  
Intermediate Compartment

The biosynthetic secretory pathway is particularly challenging to investigate as it is underrepresented compared to the abundance of the other intracellular trafficking routes. Here, we combined the retention using selective hook (RUSH) to a CRISPR-Cas9 gene editing approach (eRUSH) and identified Rab7-harboring vesicles as an important intermediate compartment of the Golgi–to–plasma membrane transport of neosynthesized transferrin receptor (TfR). These vesicles did not exhibit degradative properties and were not associated to Rab6A-harboring vesicles. Rab7A was transiently associated to neosynthetic TfR-containing post–Golgi vesicles but dissociated before fusion with the plasma membrane. Together, our study reveals a role for Rab7 in the biosynthetic secretory pathway of the TfR, highlighting the diversity of the secretory vesicles’ nature.

scholarly journals The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

2014 ◽  
Vol 25 (23) ◽  
pp. 3813-3822 ◽  
Author(s):  
Guangzuo Luo ◽  
Jian Zhang ◽  
Wei Guo
Keyword(s):  
Membrane Trafficking ◽  
Secretory Vesicle ◽  
The Other ◽  
Rab Gtpase ◽  
Secretory Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Exocyst Complex ◽  
Nucleotide Exchange Factor

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

scholarly journals The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

2016 ◽  
Vol 27 (4) ◽  
pp. 686-701 ◽  
Author(s):  
Danièle Stalder ◽  
Peter J. Novick
Keyword(s):  
Secretory Pathway ◽  
Rab Gtpase ◽  
Secretory Vesicles ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Casein Kinases ◽  
Phosphatidylinositol 4 ◽  
Acting In Concert

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling.

scholarly journals Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

2021 ◽  
Vol 22 (10) ◽  
pp. 5369
Author(s):  
Martina Pirro ◽  
Yassene Mohammed ◽  
Arnoud H. de Ru ◽  
George M. C. Janssen ◽  
Rayman T. N. Tjokrodirijo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Quantitative Proteomics ◽  
Glycosylation Site ◽  
Total Cell ◽  
Tandem Mass ◽  
Proteomics Data ◽  
Site Specific ◽  
Oxonium Ions ◽  
Tandem Mass Tag ◽  
Colorectal Cancer Cell Lines

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

scholarly journals Quantitative Proteomics Analysis Reveals the Function of the Putative Ester Cyclase UvEC1 in the Pathogenicity of the Rice False Smut Fungus Ustilaginoidea virens

2021 ◽  
Vol 22 (8) ◽  
pp. 4069
Author(s):  
Xiaoyang Chen ◽  
Zhangxin Pei ◽  
Pingping Li ◽  
Xiabing Li ◽  
Yuhang Duan ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Fungal Pathogens ◽  
Protein Localization ◽  
Proteomics Data ◽  
Ustilaginoidea Virens ◽  
Rice False Smut ◽  
Oxidative Stresses ◽  
Tandem Mass Tag ◽  
Cyclase Domain ◽  
False Smut

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

2021 ◽  
Author(s):  
Janine McCaughey ◽  
Judith M. Mantell ◽  
Chris R. Neal ◽  
Kate Heesom ◽  
David J. Stephens
Keyword(s):  
Endoplasmic Reticulum ◽  
Light Microscopy ◽  
Secretory Pathway ◽  
Genome Engineering ◽  
High Volume ◽  
Vesicle Budding ◽  
Early Secretory Pathway ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Golgi Organization

AbstractComplex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

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