scholarly journals A Cell-Specific Transgenic Approach in Xenopus Reveals the Importance of a Functional p24 System for a Secretory Cell

2004 ◽  
Vol 15 (3) ◽  
pp. 1244-1253 ◽  
Author(s):  
Gerrit Bouw ◽  
Rick Van Huizen ◽  
Eric J.R. Jansen ◽  
Gerard J.M. Martens
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Transgenic Animal ◽  
Secretory Protein ◽  
Physiological Effect ◽  
Early Secretory Pathway ◽  
Golgi Transport ◽  
Cargo Transport ◽  
Transport Vesicles ◽  
A Cell

The p24α, -β, -γ, and -δ proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24δ2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24δ2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.

scholarly journals Erv14p Directs a Transmembrane Secretory Protein into COPII-coated Transport Vesicles

2002 ◽  
Vol 13 (3) ◽  
pp. 880-891 ◽  
Author(s):  
Jacqueline Powers ◽  
Charles Barlowe
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Conserved Residues ◽  
Early Secretory Pathway ◽  
Transport Vesicles ◽  
Copii Vesicle ◽  
Copii Vesicles ◽  
Selection Of

Erv14p is a conserved integral membrane protein that traffics in COPII-coated vesicles and localizes to the early secretory pathway in yeast. Deletion of ERV14 causes a defect in polarized growth because Axl2p, a transmembrane secretory protein, accumulates in the endoplasmic reticulum and is not delivered to its site of function on the cell surface. Herein, we show that Erv14p is required for selection of Axl2p into COPII vesicles and for efficient formation of these vesicles. Erv14p binds to subunits of the COPII coat and binding depends on conserved residues in a cytoplasmically exposed loop domain of Erv14p. When mutations are introduced into this loop, an Erv14p-Axl2p complex accumulates in the endoplasmic reticulum, suggesting that Erv14p links Axl2p to the COPII coat. Based on these results and further genetic experiments, we propose Erv14p coordinates COPII vesicle formation with incorporation of specific secretory cargo.

scholarly journals Targeting CCR5 trafficking to inhibit HIV-1 infection

2019 ◽  
Vol 5 (10) ◽  
pp. eaax0821 ◽  
Author(s):  
Gaelle Boncompain ◽  
Floriane Herit ◽  
Sarah Tessier ◽  
Aurianne Lescure ◽  
Elaine Del Nery ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Molecular Level ◽  
High Content Screening ◽  
Differential Protein ◽  
Early Secretory Pathway ◽  
A Cell ◽  
Hiv Entry ◽  
Hiv 1

Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.

scholarly journals Differential Induction of Two p24δ Putative Cargo Receptors upon Activation of a Prohormoneproducing Cell

2000 ◽  
Vol 11 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Roland P. Kuiper ◽  
Hans R. Waterham ◽  
Jutta Rötter ◽  
Gerrit Bouw ◽  
Gerard J. M. Martens
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Fold Increase ◽  
Type I ◽  
Black Background ◽  
Background Adaptation ◽  
Cargo Transport ◽  
Transport Vesicles ◽  
Identification And Characterization

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24α, -β, -γ, and -δ. We previously identified a member of the p24δ subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis(∼30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24δ member (Xp24δ2) and on the identification and characterization of a second member (Xp24δ1) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24δ2. The two p24δ members are ubiquitously expressed, but Xp24δ2 is neuroendocrine enriched. During black background adaptation, the amount of the Xp24δ2 protein in the intermediate pituitary was increased ∼25 times, whereas Xp24δ1 protein expression was increased only 2.5 times. Furthermore, the level of Xp24δ2 mRNA was ∼5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24δ1 mRNA expression was not induced. Therefore, the expression of Xp24δ2specifically correlates with the expression of POMC. Together, our findings suggest that p24δ proteins have a role in selective protein transport in the secretory pathway.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

scholarly journals A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Margarita Zacharogianni ◽  
Angelica Aguilera-Gomez ◽  
Tineke Veenendaal ◽  
Jan Smout ◽  
Catherine Rabouille
Keyword(s):  
Amino Acid ◽  
Cell Viability ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Liquid Droplet ◽  
Protein Translation ◽  
Amino Acid Starvation ◽  
Early Secretory Pathway ◽  
Er Exit Sites ◽  
Nutritional Restriction

Nutritional restriction leads to protein translation attenuation that results in the storage and degradation of free mRNAs in cytoplasmic assemblies. In this study, we show in Drosophila S2 cells that amino-acid starvation also leads to the inhibition of another major anabolic pathway, the protein transport through the secretory pathway, and to the formation of a novel reversible non-membrane bound stress assembly, the Sec body that incorporates components of the ER exit sites. Sec body formation does not depend on membrane traffic in the early secretory pathway, yet requires both Sec23 and Sec24AB. Sec bodies have liquid droplet-like properties, and they act as a protective reservoir for ERES components to rebuild a functional secretory pathway after re-addition of amino-acids acting as a part of a survival mechanism. Taken together, we propose that the formation of these structures is a novel stress response mechanism to provide cell viability during and after nutrient stress.

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 Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

scholarly journals The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993 ◽  
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

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