The GTPase Arf1 Is a Determinant of Yeast Vps13 Localization to the Golgi Apparatus

VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13′s interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.

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Activation of Neurokinin 3 Receptors Stimulates GnRH Release in a Location-Dependent but Kisspeptin-Independent Manner in Adult Mice

Endocrinology ◽

10.1210/en.2013-1479 ◽

2013 ◽

Vol 154 (11) ◽

pp. 3984-3989 ◽

Cited By ~ 35

Author(s):

Garrett T. Gaskins ◽

Katarzyna M. Glanowska ◽

Suzanne M. Moenter

Keyword(s):

Green Fluorescent Protein ◽

Carbon Fiber ◽

Fluorescent Protein ◽

Hypogonadotropic Hypogonadism ◽

Brain Slices ◽

Dependent Manner ◽

Gnrh Secretion ◽

Gnrh Release ◽

Carbon Fiber Microelectrodes ◽

Green Fluorescent

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

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Internalization of Monomeric Lipopolysaccharide Occurs after Transfer Out of Cell Surface Cd14

Journal of Experimental Medicine ◽

10.1084/jem.190.4.509 ◽

1999 ◽

Vol 190 (4) ◽

pp. 509-522 ◽

Cited By ~ 58

Author(s):

Thierry Vasselon ◽

Eric Hailman ◽

Rolf Thieringer ◽

Patricia A. Detmers

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Golgi Apparatus ◽

Cell Surface ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Soluble Cd14 ◽

Stable Transfectants ◽

Intracellular Vesicles ◽

Green Fluorescent

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14–EGFP) was used to follow trafficking of mCD14 and BODIPY–LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex–like structure. mCD14–EGFP was functional in mediating binding of and responses to LPS. BODIPY–LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14–EGFP on the cell surface. However, within 5–10 min, the BODIPY–LPS distributed to intracellular vesicles that did not contain mCD14–EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

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Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

Biochemical Journal ◽

10.1042/bj3570687 ◽

2001 ◽

Vol 357 (3) ◽

pp. 687-697 ◽

Cited By ~ 21

Author(s):

Jonathan P. WAUD ◽

Alexandra BERMÚDEZ FAJARDO ◽

Thankiah SUDHAHARAN ◽

Andrew R. TRIMBY ◽

Jinny JEFFERY ◽

...

Keyword(s):

Energy Transfer ◽

Green Fluorescent Protein ◽

Caspase 3 ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dependent Manner ◽

Cell Extracts ◽

Donor And Acceptor ◽

Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

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Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

Journal of Bacteriology ◽

10.1128/jb.182.11.3254-3258.2000 ◽

2000 ◽

Vol 182 (11) ◽

pp. 3254-3258 ◽

Cited By ~ 23

Author(s):

D. K. Stafslien ◽

P. P. Cleary

Keyword(s):

Green Fluorescent Protein ◽

Fusion Protein ◽

Fluorescent Protein ◽

High Sensitivity ◽

Dependent Manner ◽

Protein Fusion ◽

Group B Streptococci ◽

Mutant Proteins ◽

Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

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Calcineurin Localizes to the Hyphal Septum in Aspergillus fumigatus: Implications for Septum Formation and Conidiophore Development

Eukaryotic Cell ◽

10.1128/ec.00200-08 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1606-1610 ◽

Cited By ~ 31

Author(s):

Praveen Rao Juvvadi ◽

Jarrod R. Fortwendel ◽

Nadthanan Pinchai ◽

B. Zachary Perfect ◽

Joseph Heitman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Aspergillus Fumigatus ◽

Filamentous Fungus ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Dependent Manner ◽

Septum Formation ◽

Calcineurin A ◽

Green Fluorescent

ABSTRACT A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus ΔcnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.

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Arfophilins Are Dual Arf/Rab 11 Binding Proteins That Regulate Recycling Endosome Distribution and Are Related to Drosophila Nuclear Fallout

Molecular Biology of the Cell ◽

10.1091/mbc.e03-03-0160 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2908-2920 ◽

Cited By ~ 95

Author(s):

Gilles R.X. Hickson ◽

Johanne Matheson ◽

Blake Riggs ◽

Valerie H. Maier ◽

Andrew B. Fielding ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Binding Capacity ◽

Binding Protein ◽

Focal Adhesions ◽

Dependent Manner ◽

Recycling Endosome ◽

Nuclear Fallout ◽

Endosomal Recycling ◽

Green Fluorescent

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.

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Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

The Journal of Cell Biology ◽

10.1083/jcb.152.5.935 ◽

2001 ◽

Vol 152 (5) ◽

pp. 935-944 ◽

Cited By ~ 102

Author(s):

Ken Sato ◽

Miyuki Sato ◽

Akihiko Nakano

Keyword(s):

Endoplasmic Reticulum ◽

Green Fluorescent Protein ◽

Membrane Proteins ◽

Golgi Apparatus ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Endoplasmic Reticulum Membrane ◽

Golgi Membrane ◽

Green Fluorescent

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

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Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells

Blood ◽

10.1182/blood-2003-01-0049 ◽

2003 ◽

Vol 102 (6) ◽

pp. 2115-2121 ◽

Cited By ~ 38

Author(s):

Fan Zhang ◽

Neil R. Hackett ◽

George Lam ◽

Joseph Cheng ◽

Robert Pergolizzi ◽

...

Keyword(s):

Endothelial Cells ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Adenoviral Delivery ◽

Mitogen Activated Protein ◽

Transfer Vectors ◽

Cox 2 ◽

Green Fluorescent

Abstract Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.

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p120-Catenin Regulates Clathrin-dependent Endocytosis of VE-Cadherin

Molecular Biology of the Cell ◽

10.1091/mbc.e05-05-0440 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5141-5151 ◽

Cited By ~ 177

Author(s):

Kanyan Xiao ◽

Jennifer Garner ◽

Kathleen M. Buckley ◽

Peter A. Vincent ◽

Christine M. Chiasson ◽

...

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Dependent Manner ◽

Lysosomal Degradation ◽

P120 Catenin ◽

Binding Partner ◽

Endocytic Machinery ◽

Green Fluorescent ◽

Retention Signal

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin–p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

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Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.23 ◽

2000 ◽

Vol 11 (1) ◽

pp. 23-38 ◽

Cited By ~ 250

Author(s):

Michael J. Lewis ◽

Benjamin J. Nichols ◽

Cristina Prescianotto-Baschong ◽

Howard Riezman ◽

Hugh R. B. Pelham

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Golgi Apparatus ◽

Cell Surface ◽

Immunoelectron Microscopy ◽

Fluorescent Protein ◽

Transmembrane Domain ◽

Early Endosomes ◽

Internal Structures ◽

Green Fluorescent

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

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