Functional characterization of Hsc70 -interacting protein (Hip) and chaperone interactions in Vitis labrusca and Arabidopsis thaliana
Abstract
Most angiosperms accumulate calcium oxalate crystals in defined patterns of crystal morphology and distribution. In grape (Vitis) needle-shaped crystals, called raphides, develop in intravacuolar membrane compartments, termed crystal chambers. We previously identified a cDNA, designated RAPS, which putatively encodes a crystal chamber protein in grape. RAP3 encodes a chime c protein similar to the coehaperone Hip (Hsc70-interacting protein) and to thioredoxin in separate domains. Within the Hip domain tetratricopeptide repeats necessary for interaction with Hsc70 are conserved. With immunoblot analysis we have detected both Hip and Hsc70 in protein samples extracted from isolated grape raphides. The Arabidopsis genome contains two genes encoding Hip. One was similar to mammalian Hip, and the second was similar to the Hip/thioredoxin chimera in grape. All known Hip proteins contain a distinct acidic domain near the N-terminus. To determine whether this domain binds calcium, we assayed Hip proteins from grape and Arabidopsis for calcium binding. In all cases we found that full-length recombinant Hip bound calcium, whereas truncated Hip from which the acidic domain was deleted did not bind calcium. Our results show for the first time that Hip is a calcium-binding protein and that the acidic domain is necessary for calcium binding. Genetic and biochemical studies were pursued in Arabidopsis to gain additional information about potential functions of Hip in plants. RT-PCR analyses have shown that both Hip genes are ubiquitously expressed in Arabidopsis. Putative null mutants were isolated for one of the two Hip genes. Under certain conditions we have observed differences in growth of mutants compared to wild-type plants. Co-immunoprecipitation experiments in Arabidopsis, using anti-Hsc70 antibody to identify Hsc70 interactions, did not reveal co-precipitation of Hip with Hsc70, but this may require defined conditions not yet identified. Hsc70 co-precipitated with numerous other proteins, including an ortholog of ycf1, an ABC transporter controlling vacuolar partitioning of ions. This might reveal a role for Hsc70 and its interacting proteins in regulating ion transport in plants.
Degree
Ph.D.
Advisors
Webb, Purdue University.
Subject Area
Cellular biology|Genetics|Molecular biology
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