Functional characterization of novel human ARFGAP3

Liu, Xiaoqin; Zhang, Chenggang; Xing, Guichun; Chen, Qingtang; He, Fuchu

doi:10.1016/s0014-5793(01)02134-2

Cited by 36 publications

(28 citation statements)

References 26 publications

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“…This fragment replaced the part that was removed from the plasmid above to generate pEGFPN3-ARFGAP2. Fulllength ARFGAP3 (NM_014570) was generated by PCR using pBAD-ARFGAP3 as a template (30) and inserted into pCMUIV using the BamHI restriction sites (5Ј and 3Ј). Plasmid encoding for ARFGAP3⌬140 -521 was generated by PCR, digested, and inserted into pRSETA and pGEX-4T-3 using BamHI restriction sites (5Ј and 3Ј).…”

Section: Methodsmentioning

confidence: 99%

“…The role of lipid regulation of Glo3-type ARFGAPs remains an open question. ARFGAP3 activity shows some sensitivity to lipids, but the functional significance of this is not known (30). The importance of lipids for ARFGAP1 binding to the Golgi is highlighted by the rapid dissociation of ARF-GAP1 upon the addition of propanolol, which prevents the formation of diacylglycerol (40).…”

Section: Arfgap2 and Arfgap3 Are An Essential Pair For Copi Coatmentioning

confidence: 99%

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ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Kartberg

Asp

Dejgaard

et al. 2010

Journal of Biological Chemistry

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Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.

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Section: Methodsmentioning

confidence: 99%

Section: Arfgap2 and Arfgap3 Are An Essential Pair For Copi Coatmentioning

confidence: 99%

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Kartberg

Asp

Dejgaard

et al. 2010

Journal of Biological Chemistry

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“…In brief, 293T cells, which secrete appreciable levels of SEAP (Pertea et al 2003), were transfected with pCMV, phRIP, phRIP⌬N360, or pEGFP in the presence of pCMV ␤-gal, and endogenous SEAP activity was monitored using a standard chromogenic enzyme assay (Liu et al 2001). To control the specificity of inhibition, we treated mock transfected cells with Brefeldin A (BFA), a fungal toxin that selectively disrupts trafficking through the Golgi (Chardin and McCormick 1999).…”

Section: Genes and Development 27mentioning

confidence: 99%

“…Cells and culture supernatants were harvested at 48 and 72 h posttransfection and SEAP activity assays were performed as described in Liu et al (2001). The level of SEAP secretion was quantified as the ratio of SEAP activity in culture supernatants to the sum of SEAP activity in both the culture supernatants and cell lysates.…”

Section: Genes and Development 31mentioning

confidence: 99%

hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region

Sánchez-Velar

Udofia²,

Yu³

et al. 2003

Genes Dev.

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Human immunodeficiency virus Rev facilitates the cytoplasmic accumulation of viral RNAs that contain aRev binding site. A human Rev-interacting protein (hRIP) was originally identified based on its ability to interact with the Rev nuclear export signal (NES) in yeast two-hybrid assays. To date, however, the function of hRIP and a role for hRIP in Rev-directed RNA export have remained elusive. Here we ablate hRIP activity with a dominant-negative mutant or RNA interference and analyze Rev function by RNA in situ hybridization. We find, unexpectedly, that in the absence of functional hRIP, Rev-directed RNAs mislocalize and aberrantly accumulate at the nuclear periphery, where hRIP is localized. In contrast, in the absence of Rev or the Rev cofactor CRM1, Rev-directed RNAs remain nuclear. We further show that the RNA mislocalization pattern resulting from loss of hRIP activity is highly specific to Rev function: the intracellular distribution of cellular poly(A) + mRNA, nuclear proteins, and, most important, NES-containing proteins, are unaffected. Thus, hRIP is an essential cellular Rev cofactor, which acts at a previously unanticipated step in HIV-1 RNA export: movement of RNAs from the nuclear periphery to the cytoplasm. Animal viruses often encode regulatory proteins, which facilitate expression of their viral genes in the host cell. One such example is the human immunodeficiency virus type 1 (HIV-1) Rev protein, which uses a novel mechanism to regulate viral messenger RNA (mRNA) export from the nucleus. Rev is a sequence-specific RNA binding protein that facilitates the cytoplasmic accumulation of the intron-containing HIV-1 gag-pol and env mRNAs (Cullen 2002). Rev interacts with a cis-acting viral RNA element in these mRNAs designated the Rev responsive element (RRE; Pollard and Malim 1998; Hope 1999). Rev contains two distinct functional domains: an arginine-rich RNA binding motif (ARM) and a leucinerich "effector" domain. Rev's ARM domain confers sequence-specific RNA binding, protein oligomerization, and nuclear targeting (Frankel and Young 1998). Rev's effector domain contains a leucine-rich nuclear export signal (NES), which binds the nuclear export receptor CRM1 (Weis 2002). Other cellular proteins have been shown to interact directly or indirectly with the Rev NES, including the kinesin-like Rev/Rex effector binding protein (REBP) and the eukaryotic initiation factor-5 alpha (eIF-5␣; Bevec et al. 1996;Dayton 1996;Kjems and Askjaer 2000;Venkatesh et al. 2003).The human Rev-interacting protein (hRIP), also known as hRab or Hrb, was identified using a yeast twohybrid screen with Rev as the bait (Bogerd et al. 1995;Fritz et al. 1995). Additional studies demonstrated a strong correlation between the ability of Rev NES mutants to support Rev function in yeast and their ability to interact with hRIP in the two-hybrid assay (Stutz et al. 1996). However, subsequent genetic analyses indicate the Rev-hRIP interaction is indirect, and is likely bridged by CRM1 (Fritz and Green 1996;Henderson and Percipalle 1997;Ne...

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“…15 and 16). Evidence for the Golgi function of the mammalian proteins includes their localization at the Golgi complex (11,13,14), their functional (17, 18) and physical (14,19,20) interaction with the COPI coat, and the perturbation of Golgi integrity upon overexpression of the ArfGAP1 protein (21).A unique feature of ArfGAP1 is its avid interaction with loosely packed lipids presented by liposomes containing diacylglycerols (22) or by small liposomes where the high curvature of the bilayer creates open spaces between the phospholipid acyl chains (23,24). The response of ArfGAP1 to membrane curvature was proposed to serve as a mechanism for the regulation of GTP hydrolysis during bud formation.…”

mentioning

confidence: 99%

Golgi Localization Determinants in ArfGAP1 and in New Tissue-specific ArfGAP1 Isoforms

Parnis

Rawet

Regev

et al. 2006

Journal of Biological Chemistry

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The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulumGolgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204 -214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244 -2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue inframe insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.Membrane traffic in the endoplasmic reticulum-Golgi shuttle is mediated by the COPI 3 and COPII trafficking systems. The COPII system mediates the initial exit from the endoplasmic reticulum, whereas subsequent transport to the Golgi apparatus and retrograde Golgi to endoplasmic reticulum traffic involves COPI carriers. The COPI and COPII coats are composed of evolutionarily distinct sets of proteins yet follow similar pathways of coat assembly and disassembly (for recent reviews see Refs. 1-3). In both systems a small GTPase (Arf1 and Sar1 for COPI and COPII, respectively) plays a key regulatory role. Following activation by a guanine nucleotide exchange protein, the GTPase translocates from cytosol to the organelle membrane, where it initiates the process of coat formation by the direct binding of coat subunits (4 -6). The coat in turn recruits cargo and polymerizes causing membrane deformation to form a bud.In the second phase of the GTPase cycle of Arf1 and Sar1, bound GTP is hydrolyzed with the aid of a GTPase-activating protein (GAP). The hydrolysis of GTP is a prerequisite for coat dissociation, and its inhibition leads to the accumulation of coated vesicles that are prevented from fusing with the target membrane (4, 7). The GAP for Sar1 is a subunit of the first layer of the COPII coat, the Sec23/24 complex (8). ArfGAPs constitute a large family of proteins containing a hi...

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Functional characterization of novel human ARFGAP3

Cited by 36 publications

References 26 publications

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region

Golgi Localization Determinants in ArfGAP1 and in New Tissue-specific ArfGAP1 Isoforms

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