An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1.
GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.
Latijnhouwers
Maita
M
Cell-to-Cell Communication Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK.
Hawes
Chris
C
Carvalho
Claudine
C
Oparka
Karl
K
Gillingham
Alison K
AK
Boevink
Petra
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics; D021601:trans-Golgi Network / Q000378:metabolism*
C479928:ARL1 protein, Arabidopsis; D029681:Arabidopsis Proteins; C496228:GRIP protein, Arabidopsis; D008565:Membrane Proteins; D020559:Monomeric GTP-Binding Proteins