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 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 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 Adaptor protein 2–mediated endocytosis of the β-secretase BACE1 is dispensable for amyloid precursor protein processing

2012 ◽  
Vol 23 (12) ◽  
pp. 2339-2351 ◽  
Author(s):  
Yogikala Prabhu ◽  
Patricia V. Burgos ◽  
Christina Schindler ◽  
Ginny G. Farías ◽  
Javier G. Magadán ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amyloid Precursor Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Amyloid Precursor Protein Processing ◽  
Intermediate Compartment ◽  
Golgi Network

The β-site amyloid precursor protein (APP)–cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease that catalyzes the proteolytic processing of APP and other plasma membrane protein precursors. BACE1 cycles between the trans-Golgi network (TGN), the plasma membrane, and endosomes by virtue of signals contained within its cytosolic C-terminal domain. One of these signals is the DXXLL-motif sequence DISLL, which controls transport between the TGN and endosomes via interaction with GGA proteins. Here we show that the DISLL sequence is embedded within a longer [DE]XXXL[LI]-motif sequence, DDISLL, which mediates internalization from the plasma membrane by interaction with the clathrin-associated, heterotetrameric adaptor protein 2 (AP-2) complex. Mutation of this signal or knockdown of either AP-2 or clathrin decreases endosomal localization and increases plasma membrane localization of BACE1. Remarkably, internalization-defective BACE1 is able to cleave an APP mutant that itself cannot be delivered to endosomes. The drug brefeldin A reversibly prevents BACE1-catalyzed APP cleavage, ruling out that this reaction occurs in the endoplasmic reticulum (ER) or ER–Golgi intermediate compartment. Taken together, these observations support the notion that BACE1 is capable of cleaving APP in late compartments of the secretory pathway.

scholarly journals Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane

2018 ◽  
Vol 29 (6) ◽  
pp. 736-750 ◽  
Author(s):  
Dongmei Liu ◽  
Xia Li ◽  
David Shen ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
The Other ◽  
Secretory Vesicles ◽  
Endocytic Recycling ◽  
C Terminus ◽  
Tm Domain

The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion. We anchored each subunit with a transmembrane (TM) domain at its N- or C-terminus. Only N-terminally anchored TM-Sec3p and C-terminally anchored Exo70p-TM proved functional. These findings orient the complex with respect to the membrane and establish that Sec3p and Exo70p can function exclusively on the membrane. The functions of TM-Sec3p and Exo70p-TM were largely unaffected by blocks in endocytic recycling, suggesting that they act on the plasma membrane rather than on secretory vesicles. Cytosolic pools of the other exocyst subunits were unaffected in TM-sec3 cells, while they were partially depleted in exo70-TM cells. Blocking actin-dependent delivery of secretory vesicles in act1-3 cells results in loss of Sec3p from the purified complex. Our results are consistent with a model in which Sec3p and Exo70p can function exclusively on the plasma membrane while the other subunits are brought to them on secretory vesicles.

scholarly journals Dominant Negative Alleles ofSEC10Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

1998 ◽  
Vol 9 (7) ◽  
pp. 1725-1739 ◽  
Author(s):  
Dagmar Roth ◽  
Wei Guo ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Cycle Progression ◽  
Vesicle Docking ◽  
Amino Terminal ◽  
Exocyst Complex ◽  
Carboxy Terminal

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeastSaccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

scholarly journals Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

2016 ◽  
Vol 113 (24) ◽  
pp. 6677-6682 ◽  
Author(s):  
Yongqiang Deng ◽  
Felix E. Rivera-Molina ◽  
Derek K. Toomre ◽  
Christopher G. Burd
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Secretory Vesicle ◽  
Secretory Protein ◽  
Secretory Vesicles ◽  
Distinct Class ◽  
Equinatoxin Ii ◽  
Golgi Network

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.

scholarly journals Ankyrin facilitates intracellular trafficking of α1-Na+-K+-ATPase in polarized cells

2008 ◽  
Vol 295 (5) ◽  
pp. C1202-C1214 ◽  
Author(s):  
Paul R. Stabach ◽  
Prasad Devarajan ◽  
Michael C. Stankewich ◽  
Serguei Bannykh ◽  
Jon S. Morrow
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Secretory Pathway ◽  
Cultured Cells ◽  
Er Retention ◽  
Hereditary Disorders ◽  
In Trans ◽  
Vesicular Stomatitis ◽  
Membrane Scaffold ◽  
Binding Sequence

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of α1-Na+-K+-ATPase facilitates the entry of α1,β1-Na+-K+-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of α1-Na+-K+-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type α1-Na+-K+-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of α1-Na+-K+-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization.

scholarly journals Promiscuity in Rab–SNARE Interactions

1999 ◽  
Vol 10 (12) ◽  
pp. 4149-4161 ◽  
Author(s):  
Eric Grote ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Snare Complex ◽  
Free Form ◽  
Rab Proteins ◽  
Secretory Vesicles ◽  
Rab Protein ◽  
Target Membrane ◽  
Low Efficiency

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in asec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab–SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab–SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

scholarly journals Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

2003 ◽  
Vol 77 (16) ◽  
pp. 9008-9019 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

scholarly journals Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells.

1994 ◽  
Vol 127 (6) ◽  
pp. 1589-1601 ◽  
Author(s):  
R E Leube ◽  
U Leimer ◽  
C Grund ◽  
W W Franke ◽  
N Harth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Secretory Pathway ◽  
Hepatitis B Surface Antigen ◽  
Fluid Phase ◽  
Distinct Type ◽  
Confocal Laser ◽  
Immunogold Label ◽  
Transmembrane Glycoprotein ◽  
Double Label

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double-immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.

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