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The GRIP Domain is a Specific Targeting Sequence for a Population of trans ‐Golgi Network Derived Tubulo‐Vesicular Carriers
Author(s) -
Brown Darren L.,
Heimann Kirsten,
Lock John,
KjerNielsen Lars,
van Vliet Catherine,
Stow Jennifer L.,
Gleeson Paul A.
Publication year - 2001
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2001.002005336.x
Subject(s) - golgi apparatus , biology , endomembrane system , microbiology and biotechnology , vesicle , green fluorescent protein , population , transport protein , fusion protein , peripheral membrane protein , fluorescence recovery after photobleaching , vesicle fusion , membrane protein , integral membrane protein , membrane , endoplasmic reticulum , biochemistry , synaptic vesicle , recombinant dna , demography , sociology , gene
Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled‐coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno‐electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)‐p230 GRIP fusion protein, we show binding specifically to a subset of membranes of the trans ‐Golgi network (TGN). Real‐time imaging of live HeLa cells revealed that the GFP‐p230 GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP‐p230 GRIP ‐transfected cells. Analysis of Golgi‐derived vesicles by sucrose gradient fractionation demonstrated that GFP‐p230 GRIP binds to a specific population of vesicles distinct from those labelled for β‐COP or γ‐adaptin. The GFP‐p230 GRIP fusion protein is recruited to the same vesicle population as full‐length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans ‐Golgi network tubulo‐vesicular carriers.