Pumping lipids with P4-ATPases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Rosa L. López-Marqués ◽  
Joost C.M. Holthuis ◽  
Thomas G. Pomorski
Keyword(s):  
Vesicle Formation ◽  
Potential Mechanism ◽  
Vesicle Budding ◽  
Vesicle Biogenesis ◽  
Phospholipid Transport ◽  
Endocytic Compartments

Abstract While accumulating evidence indicates that P4-ATPases catalyze phospholipid transport across cellular bilayers, their kinship to cation-pumping ATPases has raised fundamental questions concerning the underlying flippase mechanism. Loss of P4-ATPase function perturbs vesicle formation in late secretory and endocytic compartments. An intriguing concept is that P4-ATPases help drive vesicle budding by generating imbalances in transbilayer lipid numbers. Moreover, activation of P4-ATPases by phosphoinositides and other effectors of coat recruitment provide a potential mechanism to confine flippase activity to sites of vesicle biogenesis. These developments have raised considerable interest in understanding the mechanism, regulation and biological implications of P4-ATPase-catalyzed phospholipid transport.

scholarly journals Poliovirus Infection Transiently Increases COPII Vesicle Budding

2012 ◽  
Vol 86 (18) ◽  
pp. 9675-9682 ◽  
Author(s):  
Meg Trahey ◽  
Hyung Suk Oh ◽  
Craig E. Cameron ◽  
Jesse C. Hay
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Vesicle Formation ◽  
Vesicle Budding ◽  
Precursor Pool ◽  
Copii Vesicle ◽  
Early Increase ◽  
Vesicle Biogenesis ◽  
Intermediate Compartment ◽  
Copii Vesicles

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication “organelles” of PV (N. Y. Hsu et al., Cell 141:799–811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

scholarly journals Chemical-genetic disruption of clathrin function spares adaptor complex 3–dependent endosome vesicle biogenesis

2013 ◽  
Vol 24 (15) ◽  
pp. 2378-2388 ◽  
Author(s):  
Stephanie A. Zlatic ◽  
Emily J. Grossniklaus ◽  
Pearl V. Ryder ◽  
Gloria Salazar ◽  
Alexa L. Mattheyses ◽  
...  
Keyword(s):  
Light Chain ◽  
Pc12 Cell ◽  
Brefeldin A ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Vesicle Budding ◽  
Chemical Genetic ◽  
Vesicle Biogenesis ◽  
Divergent Behavior

A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.

scholarly journals GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis

2016 ◽  
Vol 215 (4) ◽  
pp. 499-513 ◽  
Author(s):  
Laura L. Thomas ◽  
J. Christopher Fromme
Keyword(s):  
Cross Talk ◽  
Vesicle Formation ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Direct Role ◽  
Definitive Evidence ◽  
Nucleotide Exchange Factor ◽  
Vesicle Biogenesis ◽  
Simultaneous Activation ◽  
Bona Fide

Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering.

scholarly journals Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.

1993 ◽  
Vol 120 (4) ◽  
pp. 865-875 ◽  
Author(s):  
N K Pryer ◽  
N R Salama ◽  
R Schekman ◽  
C A Kaiser
Keyword(s):  
Gel Filtration ◽  
Beta Subunits ◽  
Vesicle Formation ◽  
Multicopy Plasmid ◽  
Formation Reaction ◽  
Internal Repeat ◽  
Transport Vesicles ◽  
Vesicle Biogenesis ◽  
A Cell

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.

scholarly journals Annexin A2 promotes phagophore assembly by enhancing Atg16L+ vesicle biogenesis and homotypic fusion

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Kateryna Morozova ◽  
Sunandini Sidhar ◽  
Valerio Zolla ◽  
Cristina C. Clement ◽  
Brian Scharf ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Annexin A2 ◽  
Membrane Curvature ◽  
Early Event ◽  
Vesicle Formation ◽  
Deficient Mouse ◽  
Lipidomic Analysis ◽  
Vesicle Biogenesis ◽  
Mouse Cells ◽  
Membrane Budding

Abstract Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-deficient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 flux and dampening of macroautophagy are observed in dendritic cells from Anxa2 −/− mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.

scholarly journals Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations.

1996 ◽  
Vol 7 (7) ◽  
pp. 1043-1058 ◽  
Author(s):  
M J Elrod-Erickson ◽  
C A Kaiser
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Secretory Proteins ◽  
Vesicle Formation ◽  
Gene Products ◽  
Vesicle Budding ◽  
Golgi Transport ◽  
Transport Vesicles ◽  
Copii Vesicle

Although convergent evidence suggests that proteins destined for export from the endoplasmic reticulum (ER) are separated from resident ER proteins and are concentrated into transport vesicles, the proteins that regulate this process have remained largely unknown. In a screen for suppressors of mutations in the essential COPII gene SEC13, we identified three genes (BST1, BST2/EMP24, and BST3) that negatively regulate COPII vesicle formation, preventing the production of vesicles with defective or missing subunits. Mutations in these genes slow the secretion of some secretory proteins and cause the resident ER proteins Kar2p and Pdi1p to leak more rapidly from the ER, indicating that these genes are also required for proper discrimination between resident ER proteins and Golgi-bound cargo molecules. The BST1 and BST2/EMP24 genes code for integral membrane proteins that reside predominantly in the ER. Our data suggest that the BST gene products represent a novel class of ER proteins that link the regulation of vesicle coat assembly to cargo sorting.

scholarly journals Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.

1994 ◽  
Vol 127 (4) ◽  
pp. 915-934 ◽  
Author(s):  
H Damke ◽  
T Baba ◽  
D E Warnock ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Wild Type ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Hela Cell Lines

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

scholarly journals Several ADP-ribosylation Factor (Arf) Isoforms Support COPI Vesicle Formation

2011 ◽  
Vol 286 (41) ◽  
pp. 35634-35642 ◽  
Author(s):  
Vincent Popoff ◽  
Julian D. Langer ◽  
Ingeborg Reckmann ◽  
Andrea Hellwig ◽  
Richard A. Kahn ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Initial Step ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Vesicular Carriers ◽  
Vesicle Biogenesis ◽  
Copi Vesicle

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.

scholarly journals Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

1996 ◽  
Vol 135 (6) ◽  
pp. 1471-1483 ◽  
Author(s):  
C D Austin ◽  
D Shields
Keyword(s):  
Endocrine Cells ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Secretory Vesicle ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Vesicle Release ◽  
Vesicle Budding ◽  
Phosphatase Inhibitors ◽  
Gtp Binding

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

Vesicular transport: the core machinery of COPI recruitment and budding

2002 ◽  
Vol 115 (16) ◽  
pp. 3235-3240 ◽  
Author(s):  
Walter Nickel ◽  
Britta Brügger ◽  
Felix T. Wieland
Keyword(s):  
Vesicular Transport ◽  
Eukaryotic Cells ◽  
Vesicle Budding ◽  
The Core ◽  
New Findings ◽  
Vesicle Biogenesis ◽  
Bidirectional Transport ◽  
Membrane Bound ◽  
Copi Vesicle ◽  
Shed Light

Vesicular transport is the predominant mechanism for exchange of proteins and lipids between membrane-bound organelles in eukaryotic cells. Golgi-derived COPI-coated vesicles are involved in several vesicular transport steps, including bidirectional transport within the Golgi and recycling to the ER. Recent work has shed light on the mechanism of COPI vesicle biogenesis, in particular the machinery required for vesicle formation. The new findings have allowed us to generate a model that covers the cycle of coat recruitment, coat polymerization, vesicle budding and uncoating.

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