Redundant and Distinct Functions for Dynamin-1 and Dynamin-2 Isoforms

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

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PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells

AJP Cell Physiology ◽

10.1152/ajpcell.00212.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. C835-C844 ◽

Cited By ~ 5

Author(s):

Valeria Padovano ◽

Silvia Massari ◽

Silvia Mazzucchelli ◽

Grazia Pietrini

Keyword(s):

Excitatory Amino Acid ◽

Mdck Cells ◽

Glutamate Transporter ◽

Specific Inhibitor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Metabolic Labeling ◽

Apical Surface ◽

Recycling Endosomes ◽

Intracellular Compartments

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKCα. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKCα-labeled recycling endosomes common to apical and basolateral proteins.

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Rab11-like GTPase associates with and regulates the structure and function of the contractile vacuole system in Dictyostelium

Journal of Cell Science ◽

10.1242/jcs.114.16.3035 ◽

2001 ◽

Vol 114 (16) ◽

pp. 3035-3045 ◽

Cited By ~ 1

Author(s):

Edward Harris ◽

Kunito Yoshida ◽

James Cardelli ◽

John Bush

Keyword(s):

Proton Pump ◽

Membrane Trafficking ◽

Amino Acid Level ◽

Fluid Phase ◽

Membrane System ◽

Structure And Function ◽

Dominant Negative ◽

Contractile Vacuole ◽

And Function ◽

Mutant Cells

Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells that overexpressed a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared with wild-type cells, the dominant negative DdRab11-expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. In addition, the mutant cells enlarged, detached from surfaces and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11-expressing cells when compared with control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole.

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Targeting the GA Binding Protein β1L Isoform Does Not Perturb Lymphocyte Development and Function

Molecular and Cellular Biology ◽

10.1128/mcb.01855-07 ◽

2008 ◽

Vol 28 (13) ◽

pp. 4300-4309 ◽

Cited By ~ 10

Author(s):

Hai-Hui Xue ◽

Xuefang Jing ◽

Julie Bollenbacher-Reilley ◽

Dong-Mei Zhao ◽

Jodie S. Haring ◽

...

Keyword(s):

B Cells ◽

Target Genes ◽

Splice Variants ◽

Binding Protein ◽

Critical Role ◽

Dominant Negative ◽

T And B Cells ◽

Carboxy Terminal ◽

And Function ◽

Ga Binding Protein

ABSTRACT GA binding protein (GABP) is a ubiquitously expressed Ets family transcription factor that consists of two subunits, GABPα and GABPβ. GABPα binds to DNA, and GABPβ heterodimerizes with GABPα and possesses the ability to transactivate target genes. Our previous studies using GABPα-deficient mice revealed that GABPα is required for the development of both T and B cells. Two splice variants of GABPβ are generated from the Gabpb1 locus and differ in their carboxy-terminal lengths and sequences. The longer isoform (GABPβ1L) can homodimerize and thus form α2β2 tetramers depending on the gene context, whereas the shorter isoform (GABPβ1S) cannot. In this study, we generated mice that are deficient in GABPβ1L but that retain the expression of GABPβ1S. Surprisingly, GABPβ1L−/− mice had normal T- and B-cell development, and mature T and B cells showed normal responses to various stimuli. In contrast, targeting both GABPβ1L and GABPβ1S resulted in early embryonic lethality. Because of its incapability of forming homodimers, GABPβ1S has been suspected to have a dominant negative role in regulating GABP target genes. Our findings argue against such a possibility and rather suggest that GABPβ1S has a critical role in maintaining the transcriptional activity of the GABPα/β complex.

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GGA proteins: new players in the sorting game

Journal of Cell Science ◽

10.1242/jcs.114.19.3413 ◽

2001 ◽

Vol 114 (19) ◽

pp. 3413-3418 ◽

Cited By ~ 1

Author(s):

Annette L. Boman

Keyword(s):

Membrane Trafficking ◽

Sequence Homology ◽

Mammalian Cells ◽

Gene Knockout ◽

Protein Domains ◽

Vesicle Formation ◽

Trans Golgi Network ◽

And Function ◽

Golgi Network ◽

Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

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Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function

Blood ◽

10.1182/blood.v99.3.978 ◽

2002 ◽

Vol 99 (3) ◽

pp. 978-984 ◽

Cited By ~ 54

Author(s):

Jaime Millán ◽

Marı́a C. Montoya ◽

David Sancho ◽

Francisco Sánchez-Madrid ◽

Miguel A. Alonso

Keyword(s):

Lipid Rafts ◽

Mdck Cells ◽

Protein Composition ◽

Leading Edge ◽

Specific Protein ◽

Intercellular Adhesion Molecule ◽

Apical Surface ◽

Influenza Virus Hemagglutinin ◽

And Function ◽

T Lymphoblasts

Abstract Polarized migrating T cells possess 2 poles, the uropod protrusion at the rear and the leading edge at the front, with specific protein composition and function. The influenza virus hemagglutinin (HA) is a prototypical molecule that uses lipid rafts for biosynthetic transport to the apical surface in polarized epithelial Madin-Darby canine kidney (MDCK) cells. In this study, HA was used as a tool to investigate the role of lipid rafts in vectorial protein traffic in polarized T lymphocytes. Results show that newly synthesized HA becomes selectively targeted to the uropod subdomain in polarized T lymphoblasts. HA incorporates into rafts soon after biosynthesis, suggesting that delivery of HA to the uropod occurs through a pathway of transport reminiscent of that used for its specific targeting to the apical surface. HA and the adhesion molecules, intercellular adhesion molecule 3 (ICAM-3), CD44, and CD43, 3 endogenous uropod markers, were detected in surface rafts of T lymphoblasts. Cholesterol, a major component of lipid rafts, was predominantly located in the uropod. Disruption of lipid raft integrity by cholesterol sequestration produced unclustering of ICAM-3 and the loss of uropodia and severely impaired processes that require a polarized phenotype such as intercellular aggregation and cell migration. Collectively, these results indicate that lipid rafts constitute a route for selective targeting of proteins to the uropod and that the rafts are essential for the generation, maintenance, and functionality of T-cell anteroposterior polarity.

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Dynamin GTPase Domain Mutants Block Endocytic Vesicle Formation at Morphologically Distinct Stages

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2578 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2578-2589 ◽

Cited By ~ 135

Author(s):

Hanna Damke ◽

Derk D. Binns ◽

Hideho Ueda ◽

Sandra L. Schmid ◽

Takeshi Baba

Keyword(s):

Dominant Negative ◽

Coated Vesicle ◽

Vesicle Formation ◽

Complex Relationship ◽

Protein Activity ◽

Coated Pits ◽

Gtpase Domain ◽

Receptor Mediated Endocytosis ◽

Gtpase Cycle

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.

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Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

The Journal of Cell Biology ◽

10.1083/jcb.201408002 ◽

2014 ◽

Vol 207 (2) ◽

pp. 269-282 ◽

Cited By ~ 62

Author(s):

Michael Taschner ◽

Fruzsina Kotsis ◽

Philipp Braeuer ◽

E. Wolfgang Kuehn ◽

Esben Lorentzen

Keyword(s):

Crystal Structures ◽

Molecular Motors ◽

Mdck Cells ◽

Intraflagellar Transport ◽

Dominant Negative ◽

Tetratricopeptide Repeat ◽

Core Complex ◽

Disease Protein ◽

In Trans ◽

And Function

Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281–329 in a complex that interacts directly with IFT70/IFT52330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.

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