Rab8 as a Molecular Model of Vesicular Trafficking to Investigate the Latter Steps of the Secretory Pathway in Entamoeba histolytica

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

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Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010661117 ◽

2020 ◽

Vol 117 (36) ◽

pp. 22061-22067

Author(s):

Marian Samuel Vogt ◽

Gesa Felicitas Schmitz ◽

Daniel Varón Silva ◽

Hans-Ulrich Mösch ◽

Lars-Oliver Essen

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Secretory Pathway ◽

Molecular Model ◽

Md Simulations ◽

Binding Pocket ◽

Fungal Cell ◽

Final Destination ◽

Fragment Screening

The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.

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Molecular Genetics of the Secretory Pathway in Entamoeba histolytica

Archives of Medical Research ◽

10.1016/s0188-4409(00)00108-9 ◽

2000 ◽

Vol 31 (4) ◽

pp. S151-S152 ◽

Cited By ~ 2

Author(s):

Rosana Sánchez-López ◽

Angeles Gutiérrez ◽

Patricia Juárez ◽

Alejandro Olvera ◽

Felipe Olvera ◽

...

Keyword(s):

Molecular Genetics ◽

Entamoeba Histolytica ◽

Secretory Pathway

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GRASP55: A Multifunctional Protein

Current Protein and Peptide Science ◽

10.2174/1389203721666200218105302 ◽

2020 ◽

Vol 21 (6) ◽

pp. 544-552 ◽

Cited By ~ 4

Author(s):

Hongrong Wu ◽

Tianjiao Li ◽

Jianfeng Zhao

Keyword(s):

Crucial Role ◽

Protein Modification ◽

Secretory Pathway ◽

Cell Activity ◽

Vesicular Trafficking ◽

Multifunctional Protein ◽

Autophagosome Maturation

GRASP55 was first found as Golgi cisternae stacking protein. Due to the crucial role of Golgi in vesicular trafficking and protein modification, GRASP55 was found to function in these two aspects. Further investigation revealed that GRASP55 also participates in the unconventional secretory pathway under stress. Moreover, GRASP55 is involved in autophagy initiation and autophagosome maturation, as well as cell activity.

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Exocytosis in excitable cells: a conserved molecular machinery from yeast to neuron

Acta Endocrinologica ◽

10.1530/eje.0.1370001 ◽

1997 ◽

pp. 1-9 ◽

Cited By ~ 13

Author(s):

PM Lledo

Keyword(s):

Endocrine Cells ◽

Secretory Pathway ◽

Cell Types ◽

Vesicular Trafficking ◽

Cellular Functions ◽

Excitable Cells ◽

Cellular Mechanisms ◽

Molecular Machinery ◽

Intracellular Vesicles ◽

Insight Into

One of the basic cellular functions of nearly every cell type is the exocytotic release of synthesized molecules, stored and packaged into intracellular vesicles or granules. A variety of approaches has been used to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway. The findings obtained with these approaches suggest that common mechanisms may underlie a wide variety of vesicle-mediated transport steps. This review presents some of the recent findings regarding the study of the cellular mechanisms which control neurotransmitter and hormone release from neurons and endocrine cells respectively, and focuses on regulation of these mechanisms. The similarities between these two cell types can be seen as evidence to support the hypothesis according to which the regulated exocytosis apparatus could have evolved from a constitutive fusion machinery to which some key modulators have been added. Insight into secretory vesicles will be relevant not only to the understanding of vesicular trafficking or cell polarity but also to the understanding of higher nervous functions resulting from synaptic plasticity.

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Characterization of the Ehrab8 gene, a marker of the late stages of the secretory pathway of Entamoeba histolytica

Molecular and Biochemical Parasitology ◽

10.1016/s0166-6851(01)00311-5 ◽

2001 ◽

Vol 116 (2) ◽

pp. 223-228 ◽

Cited By ~ 21

Author(s):

Patricia Juárez ◽

Rosana Sanchez-Lopez ◽

Roberto P. Stock ◽

Alejandro Olvera ◽

Marco A. Ramos ◽

...

Keyword(s):

Entamoeba Histolytica ◽

Secretory Pathway ◽

Late Stages

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Vesicular Trafficking in Entamoeba histolytica is Essential for its Virulence

Eukaryome Impact on Human Intestine Homeostasis and Mucosal Immunology ◽

10.1007/978-3-030-44826-4_20 ◽

2020 ◽

pp. 275-290

Author(s):

E. Orozco ◽

A. Betanzos ◽

C. Bañuelos ◽

R. Javier-Reyna ◽

G. García-Rivera

Keyword(s):

Entamoeba Histolytica ◽

Vesicular Trafficking

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Entamoeba histolytica: intracellular distribution of the sec61α subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids

Experimental Parasitology ◽

10.1016/j.exppara.2004.12.011 ◽

2005 ◽

Vol 109 (4) ◽

pp. 241-251 ◽

Cited By ~ 9

Author(s):

Ricardo Sánchez ◽

Andrés Saralegui ◽

Alfonso Olivos-García ◽

Carlo Scapolla ◽

Gianluca Damonte ◽

...

Keyword(s):

Nucleic Acids ◽

Entamoeba Histolytica ◽

Secretory Pathway ◽

Peptide Nucleic Acids ◽

Intracellular Distribution ◽

Down Regulation ◽

Antisense Peptide

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Protein Sorting in Plasmodium Falciparum

Life ◽

10.3390/life11090937 ◽

2021 ◽

Vol 11 (9) ◽

pp. 937

Author(s):

D.C. Ghislaine Mayer

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Protein Sorting ◽

Vesicular Trafficking ◽

Unicellular Eukaryote ◽

Dense Granules ◽

Host Cell Proteins ◽

Endosymbiotic Organelle

Plasmodium falciparum is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. P. falciparum, like its relative T. gondii, uses the endolysosomal system to produce the secretory organelles and to ingest host cell proteins. The parasite also has an apicoplast, a secondary endosymbiotic organelle, which depends on vesicular trafficking for appropriate incorporation of nuclear-encoded proteins into the apicoplast. Recently, the central molecules responsible for sorting and trafficking in P. falciparum and T. gondii have been characterized. From these studies, it is now evident that P. falciparum has repurposed the molecules of the endosomal system to the secretory pathway. Additionally, the sorting and vesicular trafficking mechanism seem to be conserved among apicomplexans. This review described the most recent findings on the molecular mechanisms of protein sorting and vesicular trafficking in P. falciparum and revealed that P. falciparum has an amazing secretory machinery that has been cleverly modified to its intracellular lifestyle.

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