The GRIP Domain is a Specific Targeting Sequence for a Population of trans‐Golgi Network Derived Tubulo‐Vesicular Carriers

Abstract

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 immunoelectron 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 GRIPtransfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFPp230 GRIP binds to a specific population of vesicles distinct from those labelled for b-COP or g-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 tubulovesicular carriers.

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

“…The GRIP domain comprises ϳ42 amino acids and is shared by a family of resident peripheral Golgi coiled-coil membrane proteins, including p230, golgin-97, and golgin-245 (34). The GRIP docking site has been identified on a specific subset of Golgi membranes in the trans-Golgi network (55). Targeting eNOS to the cis/medialGolgi complex (35) with the truncated transmembrane domain of syntaxin-3 (Syn17-eNOS) produced contrasting results.…”

A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre

“…Although the N-terminus of SLMAP may serve a crucial role in mediating SLMAP-centrosome associations, the preservation of the overall structure of SLMAP may constitute an additional determinant for ensuring faithful targeting at the MTOC. Conserved motifs for protein targeting to and retention within organelles such as the ER and Golgi have been characterized (Pelham, 1988;Jackson et al, 1990;Kjer-Nielsen et al, 1999;Munro and Nichols, 1999;Brown et al, 2001), yet little is known of conserved sequences directing proteins to centrosomes. Recent studies have highlighted a conserved region of approximately 90 amino acids encoded in pericentrin and AKAP450 that confers targeting to centrosomes (Gillingham and Munro, 2000).…”

Membrane Organization and Dynamics in Cell Polarity

“…In mammalian cells, although the exocyst mostly localizes to internal membrane compartments such as Golgi and recycling endosomes Ang et al 2003;Fölsch et al 2003;Oztan et al 2007), it is recruited to designated regions of the plasma membrane where active exocytosis and membrane expansion occur. For example, in neurons undergoing differentiation or synapse formation, the exocyst is enriched at the expanding neurites and discrete puncta along axons where synapses are forming (Hsu et al 1996;Kee et al 1997;Hazuka et al 1999;Vega and Hsu, 2001;Brown et al 2001;Murthy et al 2003). In migrating cells, the exocyst is recruited to the plasma membrane at the leading edge (Zuo et al 2006;Rosse et al 2006).…”