Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, Mesembryanthemum crystallinum.
McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydrationstress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin super-family that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTIl1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton.
Chehab
E Wassim
EW
Department of Biochemistry and Molecular Biology/MS200, 311 Fleischmann Agriculture, University of Nevada, Reno, NV 89557-0014, USA.
Patharkar
O Rahul
OR
Cushman
John C
JC
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006813:Humidity; D008164:Luminescent Proteins / Q000235:genetics; D035723:Mesembryanthemum / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050766:Qb-SNARE Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D021601:trans-Golgi Network / Q000378:metabolism*
D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; D050766:Qb-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C413663:fluorescent protein 583; C120751:vesicle transport v-SNARE 11 protein, Arabidopsis; D049452:Green Fluorescent Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases