Heat Shock Protein Cognate 70-4 and an E3 Ubiquitin Ligase, CHIP, Mediate Plastid-Destined Precursor Degradation through the Ubiquitin-26S Proteasome System inArabidopsis   

Abstract

Plastid-targeted proteins pass through the cytosol as unfolded precursors. If proteins accumulate in the cytosol, they can form nonspecific aggregates that cause severe cellular damage. Here, we demonstrate that high levels of plastid precursors are degraded through the ubiquitin-proteasome system (UPS) in Arabidopsis thaliana cells. The cytosolic heat shock protein cognate 70-4 (Hsc70-4) and E3 ligase carboxy terminus of Hsc70-interacting protein (CHIP) were highly induced in plastid protein import2 plants, which had a T-DNA insertion at Toc159 and showed an albino phenotype and a severe defect in protein import into chloroplasts. Hsc70-4 and CHIP together mediated plastid precursor degradation when import-defective chloroplast-targeted reporter proteins were transiently expressed in protoplasts. Hsc70-4 recognized specific sequence motifs in transit peptides and thereby led to precursor degradation through the UPS. CHIP, which interacted with Hsc70-4, functioned as an E3 ligase in the Hsc70-4-mediated protein degradation. The physiological role of Hsc70-4 was confirmed by analyzing Hsc70-4 RNA interfernce plants in an hsc70-1 mutant background. Plants with lower Hsc70 levels exhibited abnormal embryogenesis, resulting in defective seedlings that displayed high levels of reactive oxygen species and monoubiquitinated Lhcb4 precursors. We propose that Hsc70-4 and CHIP mediate plastid-destined precursor degradation to prevent cytosolic precursor accumulation and thereby play a critical role in embryogenesis. INTRODUCTIONProteins destined for the two endosymbiotic organelles (i.e., plastids and mitochondria) are targeted from the cytoplasm as unfolded precursors (Keegstra and Froehlich, 1999;Koumoto et al., 2001;Jarvis and Soll, 2002;Soll and Schleiff, 2004;Jarvis, 2008). In the cytosol, unfolded proteins have a high tendency to form cytotoxic, life-threatening, and nonspecific aggregates if they accumulate to high levels (Wickner et al., 1999;Esser et al., 2004;Meredith, 2005;Kabashi and Durham, 2006). Therefore, posttranslational targeting to endosymbiotic organelles requires that precursor levels be maintained within limits that do not result in nonspecific aggregate formation. At the same time, the cytosolic regulatory mechanism must not jeopardize the supply of sufficient amounts of proteins to the organelles.Eukaryotic cells have a protein quality control (PQC) mechanism to constantly monitor the quality of newly synthesized proteins and preexisting proteins and to actively remove unfolded or misfolded proteins (Hartl and Hayer-Hartl, 2002;Hatakeyama and Nakayama, 2003;Esser et al., 2004). It is reported that as much as 30% of newly synthesized proteins are immediately degraded by the PQC system because of a problem in protein folding (Schubert et al., 2000). The PQC in the cytosol is achieved by two opposing processes: chaperone-assisted folding and ubiquitin/proteasome-mediated degradation. The molecular chaperones heat shock protein 70 (Hsp70) and heat shock protein cognate 70 (Hsc70), whose levels are ele...

Plastid Proteome Assembly without Toc159: Photosynthetic Protein Import and Accumulation of N-Acetylated Plastid Precursor Proteins

“…Of these genes, 1998 (8.2%) were significantly upregulated in the wild type and 2268 (9.3%) in ppi2 ( Figure 3A; see Supplemental Data Set 4 online). Our transcriptomics data confirmed the trend that photosynthetic proteins are expressed at lower levels in the import mutant as previously reported in published microarray, SAGE and RT-PCR data (Kubis et al, 2003;Kakizaki et al, 2009;Lee et al, 2009b). Notably, 84% of the downregulated genes reported in the SAGE data set were also found downregulated under our experimental conditions (see Supplemental Data Set 4 online).…”

“…This suggests that these nonphotosynthetic proteins are integrated in a signaling circuit that is different from the signaling circuit that controls the expression of photosynthetic proteins. Together, our data support the view that the accumulation of nuclear-encoded plastid precursor proteins triggers an unimported precursor protein response (Kakizaki et al, 2009;Lee et al, 2009b). This signaling response could act through additional unknown signaling pathways that do not depend on the functional state of the plastids.…”

“…ubiquitin-dependent degradation pathway for nonimported Lhcb4 precursors that is mediated by the heat shock cognate protein 70-4 and ubiquitination by an E3 ligase (Lee et al, 2009b). Inhibition of the proteasome resulted in the enhanced accumulation of ubiquitinated Lhcb4 precursors in ppi2, thus indicating the existence of a degradation system for plastid precursors.…”

The E3 Ligase TaSAP5 Alters Drought Stress Responses by Promoting the Degradation of DRIP Proteins

“…We are currently conducting yeast twohybrid assays to find possible substrates or protein partners of TaSAP5 and have identified SGT1 and HSC70 as potential interacting proteins. SGT1 was associated with components of the Skp1-Cullin-F-box protein complex in yeast and plants (Kitagawa et al, 1999;Liu et al, 2002), and HSC70 functions as a cochaperone that interacts with unfolded and denatured proteins, which are then subjected to degradation mediated by E3 ligases located in the endoplasmic reticulum (Lee et al, 2009b;Liu and Howell, 2010). Overall, TaSAP5 functioned with cooperative components by catalyzing the transfer of ubiquitin to substrate, but further research will be needed to identify the factors that enhance the protein interaction and polyubiquitin chain assembly in vivo.…”

The Ubiquitination Machinery of the Ubiquitin System

“…CHIP co-immunoprecipitates with Hsc70 after expression in protoplasts, as do the recombinant proteins (Lee et al, 2009). Hsc70 interacts with transit peptides of proteins destined to the plastid.…”