scholarly journals The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network

2001 ◽  
Vol 155 (7) ◽  
pp. 1239-1250 ◽  
Author(s):  
Pak Phi Poon ◽  
Steven F. Nothwehr ◽  
Richard A. Singer ◽  
Gerald C. Johnston
Keyword(s):  
Gtp Hydrolysis ◽  
Transport Pathways ◽  
Intracellular Vesicle ◽  
Immunofluorescence Studies ◽  
Endosomal Transport ◽  
Essential Function ◽  
Golgi Network ◽  
Intracellular Vesicle Transport ◽  
Mutant Cells ◽  
Adp Ribosylation Factor

Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

scholarly journals The Morphology of Hydroxyapatite Nanoparticles Regulates Cargo Recognition in Clathrin-Mediated Endocytosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Cheng Zhu ◽  
Xuejie Zhou ◽  
Ziteng Liu ◽  
Hongwei Chen ◽  
Hongfeng Wu ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Tumor Metastasis ◽  
High Efficiency ◽  
Therapeutic Effects ◽  
Transport Pathways ◽  
Intracellular Vesicle ◽  
Anti Cancer ◽  
Clathrin Adaptor ◽  
Intracellular Vesicle Transport ◽  
Inhibit Tumor Metastasis

The clathrin-associated protein adaptin-2 (AP2) is a distinctive member of the hetero-tetrameric clathrin adaptor complex family. It plays a crucial role in many intracellular vesicle transport pathways. The hydroxyapatite (HAp) nanoparticles can enter cells through clathrin-dependent endocytosis, induce apoptosis, and ultimately inhibit tumor metastasis. Exploring the micro process of the binding of AP2 and HAp is of great significance for understanding the molecular mechanism of HAp’s anti-cancer ability. In this work, we used molecular modeling to study the binding of spherical, rod-shaped, and needle-shaped HAps toward AP2 protein at the atomic level and found that different nanoparticles’ morphology can determine their binding specificity through electrostatic interactions. Our results show that globular HAp significantly changes AP2 protein conformation, while needle-shaped HAP has more substantial binding energy with AP2. Therefore, this work offers a microscopic picture for cargo recognition in clathrin-mediated endocytosis, clarifies the design principles and possible mechanisms of high-efficiency nano-biomaterials, and provides a basis for their potential anti-tumor therapeutic effects.

Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation

2005 ◽  
Vol 33 (4) ◽  
pp. 619-622 ◽  
Author(s):  
B. Antonny ◽  
J. Bigay ◽  
J.-F. Casella ◽  
G. Drin ◽  
B. Mesmin ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
Gtp Hydrolysis ◽  
Membrane Fission ◽  
Transport Vesicles ◽  
Highly Sensitive ◽  
Copi Vesicle ◽  
Membrane Budding ◽  
Gtpase Cycle ◽  
Adp Ribosylation Factor

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1–COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.

Molecular Cloning and Structure Analysis of MpARF, an ADP-Ribosylation Factor in Monascus purpureus

2014 ◽  
Vol 884-885 ◽  
pp. 574-577
Author(s):  
Lan Gao ◽  
Bi Yun Zhu
Keyword(s):  
Homo Sapiens ◽  
Three Dimensional ◽  
Monascus Purpureus ◽  
Protein Fold ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Gtp Hydrolysis ◽  
Adp Ribosylation ◽  
Vesicular Membrane ◽  
Adp Ribosylation Factor

ADP-ribosylation factors (ARF) are ubiquitous regulators of vesicular membrane traffic in all eukaryotic cells. A full-length cDNA encoding an ARF was cloned from the cDNA library ofMonascus purpureus. The cDNA was 1275 bp in length, contains a predicted 555 bp ORF that encodes 184 amino acids, the gene was designated MpARF. The deduced amino acid sequence showed high homology to ARF6 ofHomo sapiens, ARFB ofAspergillus nidulansand ARF3p ofSaccharomyces cerevisiae, including conserved N-terminal myristoylation site, GTP binding and GTP hydrolysis site, suggesting that the MpARF is a member of the ARF6 protein family. A typical G protein fold three-dimensional model of MpARF was built; the structure is similar to the structure of human ARF6. According to the functions of ARFB and ARF3p in fungi, we implicated that the MpARF would involved in hyphal polarized growth.

scholarly journals Molecular cloning of the mouse homologue of Rab3c

2001 ◽  
Vol 27 (1) ◽  
pp. 117-122 ◽  
Author(s):  
NJ Pavlos ◽  
J Xu ◽  
JM Papadimitriou ◽  
MH Zheng
Keyword(s):  
Skeletal Development ◽  
In Situ Hybridisation ◽  
Matrix Vesicles ◽  
Matrix Vesicle ◽  
Predicted Amino Acid Sequence ◽  
Multiple Sequence ◽  
Intracellular Vesicle ◽  
Intracellular Vesicle Transport ◽  
The Brain

Small GTP-binding proteins of the Rab subfamily are key regulators of intracellular vesicle transport. Here we report the isolation of a cDNA clone encoding the complete Rab3c isoform from mouse embryo using a degenerative PCR-based approach. Multiple sequence alignment revealed that the predicted amino acid sequence was identical to the previously identified rat Rab3c isoform and 98% identical to the published bovine Rab3c GTPase from brain. Furthermore by in situ hybridisation, Rab3c mRNA was detectable within various regions of the brain, cartilage and highly enriched within intestinal villi of foetal tissues. Chondrocytes in the hypertrophic zone, but not reserve or proliferative zones, expressed high levels of Rab3c. This pattern of expression corresponds with the genesis of matrix vesicles during endochondral ossification.In all, our results suggest that in addition to its functional role during regulated secretion in brain, Rab3c may play a part in matrix vesicle trafficking during skeletal development.

scholarly journals Analysis of the role of p200-containing vesicles in post-Golgi traffic.

1996 ◽  
Vol 7 (6) ◽  
pp. 961-974 ◽  
Author(s):  
E Ikonen ◽  
R G Parton ◽  
F Lafont ◽  
K Simons
Keyword(s):  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Apical Surface ◽  
Transport Pathways ◽  
Permeabilized Cells ◽  
Influenza Hemagglutinin ◽  
Streptolysin O ◽  
Marker Proteins ◽  
Viral Marker ◽  
Golgi Network

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.

scholarly journals Autophagy and the cvt Pathway Both Depend onAUT9

2000 ◽  
Vol 182 (8) ◽  
pp. 2125-2133 ◽  
Author(s):  
Thomas Lang ◽  
Steffen Reiche ◽  
Michael Straub ◽  
Monika Bredschneider ◽  
Michael Thumm
Keyword(s):  
Protein Turnover ◽  
Protein Transport ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Integral Membrane Protein ◽  
Biologically Active ◽  
Transport Pathways ◽  
Rich Media ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen foraut − mutants and isolated aut9-1cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic withCVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.

scholarly journals GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

1998 ◽  
Vol 18 (2) ◽  
pp. 827-838 ◽  
Author(s):  
Celeste J. Richardson ◽  
Sara Jones ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Membrane Fusion ◽  
Vesicular Transport ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Dominant Phenotype ◽  
Guanine Nucleotide Exchange ◽  
Membrane Morphology ◽  
Nucleotide Exchange Factor ◽  
Mutant Cells

ABSTRACT GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-freeYPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

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