RESUMO
J-domain proteins (JDPs) partner with Hsp70s to oversee proper synthesis, folding, transport and turnover of proteins in the cell. In any subcellular compartment, often multiple JDPs collaborate with a single Hsp70 to perform a variety of functions. Being co-localized, JDPs may exhibit complex genetic and physical interactions with each other, their clients as well as the Hsp70 partners. Even though most JDPs are highly specialized, redundancy between them is possible, making their functional analysis challenging. In the absence of assayable deletion phenotypes, protein overexpression appears to be a powerful alternative strategy to study JDP function. Here, we show that high levels of Caj1, one of the cytosolic JDPs, cause filamentous growth and G2/M arrest in yeast cells. Mutation in the critical HPD motif in the J-domain of Caj1 completely abolished these phenotypes, suggesting that Hsp70 co-chaperone function is important for the dominant-negative phenotypes exhibited by Caj1 overexpression. In this paper, we discuss the possible underlying mechanisms responsible for the pleiotropic phenotypes displayed by Caj1 overexpression in the light of current models proposed for dosage-sensitive genes (DSGs). Finally, we present generalized mechanisms of JDP overexpression-mediated dominant-negative phenotypes in budding yeast.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Apoptose/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação Fúngica da Expressão Gênica/genética , Mutação/genética , Saccharomycetales/genéticaRESUMO
Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Evolução Biológica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Sequência de Aminoácidos , Arabidopsis/classificação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosol/metabolismo , Evolução Molecular , Duplicação Gênica , Proteínas de Choque Térmico HSP70/química , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Filogenia , Conformação ProteicaRESUMO
J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.
