Abstract
ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203-334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Idelta (CKIdelta), or when cytosol was treated with antibody to CKIdelta. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIdelta also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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ADP-Ribosylation Factors / genetics
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ADP-Ribosylation Factors / metabolism*
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Animals
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CHO Cells
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COS Cells
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Casein Kinases
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Cell Membrane / metabolism*
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Cricetinae
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism*
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Golgi Apparatus / metabolism
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Green Fluorescent Proteins
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Hemagglutinins, Viral / metabolism
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Indoles / metabolism
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Isoenzymes / metabolism
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Membrane Glycoproteins / metabolism
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Phloroglucinol / analogs & derivatives*
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Phloroglucinol / metabolism
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Phosphorylation
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Protein Binding
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Protein Kinase Inhibitors
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Protein Kinases / metabolism*
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Protein Transport / physiology
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Time Factors
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Viral Envelope Proteins / metabolism
Substances
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G protein, vesicular stomatitis virus
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GTPase-Activating Proteins
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Hemagglutinins, Viral
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IC 261
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Indoles
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Isoenzymes
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Luminescent Proteins
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Membrane Glycoproteins
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Protein Kinase Inhibitors
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Recombinant Fusion Proteins
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Viral Envelope Proteins
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Green Fluorescent Proteins
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Phloroglucinol
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Protein Kinases
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Casein Kinases
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ADP-Ribosylation Factors