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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

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Abstract

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydration-stress 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 superfamily 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 AtVTI1a, 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.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

ABA:

Abscisic acid

IPTG:

Isopropyl thio-β-d-galactoside

3-AT:

3-Amino-1,2,4-triazole

Ni-NTA:

Nickel-nitrilotriacetic acid

PVC:

Prevacuolar compartment

PVDF:

Polyvinylidene fluoride

TGN:

trans-Golgi network

v-SNARE:

vesicle-soluble N-ethyl maleimide-sensitive factor attachment protein receptor

YTH:

Yeast two-hybrid

6 × His-tag:

Six histidine tag

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Acknowledgments

The authors would like to thank Dr. Tahar Taybi for providing the McCPK1–GFP construct. We are grateful to Dr. Phillip James for providing us with the yeast host stain, PJ69-4A, which was used for this work. We also thank Jen Sheen (Massachusetts General Hospital, Boston, MA, USA) for kindly providing the 35S-GFP-TYG-nos and HBT95 constructs, Dr. Jeff Harper (University of Nevada, Reno) for kindly providing the GFP-KSRM construct, and Inhan Hwang (Gyeongsang National University, Korea) for kindly providing AtVTI1a-GFP and BiP-GFP constructs. The authors would also like to thank Joan Rowe at the University of Nevada Genomics Center for automated DNA sequencing services. This work was supported in part by the U.S. Department of Agriculture - National Research Initiative - Competitive Grants Program (Grant No. 98-35100-6035), by the National Science Foundation (Grant No. MCB-0114769), and the Nevada Agricultural Experiment Station, and is published as publication No. 03066952 of the University of Nevada Agricultural Experiment Station.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology/MS200, 311 Fleischmann Agriculture, University of Nevada, Reno, NV, 89557-0014, USA

    John C. Cushman

  2. Section of Plant Biology, 1203 Life Sciences, University of California-Davis, One Shields Ave., Davis, CA, 95616-5270, USA

    E. Wassim Chehab

  3. Department of Molecular Biology, Wellman 11, Massachusetts General Hospital, Boston, MA, 02114, USA

    O. Rahul Patharkar

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  1. E. Wassim Chehab
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  2. O. Rahul Patharkar
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  3. John C. Cushman
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Correspondence to John C. Cushman.

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Chehab, E.W., Patharkar, O.R. & Cushman, J.C. 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 . Planta 225, 783–799 (2007). https://doi.org/10.1007/s00425-006-0371-4

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