The Identification of a Heat-Shock Protein Complex in Chloroplasts of Barley Leaves

Abstract

In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C3 species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This chaperone molecule does not take part in the final assembled complex (31). One such molecular chaperone within the chloroplast is RBP, which mediates the correct assembly of the Rubisco holoenzyme (9, 19). Despite the high degree of sequence homology between RBP and HSP60 proteins, however, it has yet to be shown that the synthesis of RBP increases significantly during heat shock. In plant cells experiencing heat stress, many of the induced cytoplasmic HSPs aggregate into morphologically unique structures known as HSGs (21,22,[25][26][27]. HSGs appear in the cytoplasm as dense granular complexes under the electron microscope (21, 27) but exist as smaller-sized precursor particles under normal conditions. They consist of protein and RNA, with the protein component consisting of 5 to 10% of total cytosolic HSP70 and up to 80% of all HSP20 proteins synthesized during heat shock (22,25). The RNA portion consists of non-heat-shock mRNA transcribed prior to high temperatures, and heat-shock mRNA synthesized at the onset of heat stress (22,27). HSGs are thought to function as transient storage sites for non-heat-shock mRNA, preventing their degradation during heat stress (27). When temperatures are lowered after a period of heat shock, the HSGs become more dispersed and associate closely with organelles active in protein synthesis, such as polysomes. This facilitates the rapid reactivation of control mRNA translation during recovery (21).Similar riboprotein complexes occur in the nucleolus as large perichromatin granules (21,22,24,33). These HSGs contain unprocessed precursor RNA species due to the thermal inactivation of intron splicing. Like cytoplasmic HSGs, the perichromatin granules disintegrate upon recovery and the return of normal nucleolus ultrastructure and functional processing of immature RNA (24,33).As yet, no comparable HSG structure or heat-inducible molecular chaperone has been observed in chloroplasts during heat shock. A recent study, however, has reported the formation of a HSP complex (200 kD) in the stroma of pea chloroplasts (2) consisting of a well-characterized 21-kD HSP (7,12,37,39,40). This study proposed that the 200-kD complex was the functional form of the 21-kD HSP in vivo, suggesting a possible homology to the cytoplasmic HSGs (2).The aim of this study was first to identify the range of HSPs synthesi...

Two functionally distinct forms of the photosystem II reaction-center protein D1 in the cyanobacterium Synechococcus sp. PCC 7942.

“…Proteins were transferred electrophoretically to nitrocellulose and immunoblot analysis was performed (16). Density scanning of immunoblots was carried out with a DU-8 spectrophotometer (Beckman) according to the manufacturer's recommendations.…”

Structural and Functional Insights into the Chloroplast ATP-Dependent Clp Protease in Arabidopsis

“…Stromal proteins were concentrated (Microsep 10K omega biomolecular separation kit; Pall), and protein concentrations were determined using the BCA protein assay according to the manufacturer's protocol (Pierce Chemical). Protein samples from wild-type Arabidopsis and clpP6 antisense lines (60 mg) were loaded on 4 to 13% polyacrylamide Tris-borate gels (45 mM Tris and 45 mM boric acid, pH 8.3) and electrophoresed at 20 mA for 16 h, conditions in which the molecular mass standards had reached their pore limitation for more accurate size determination of native protein complexes (Clarke and Critchley, 1992). Molecular mass standards were ferritin (440-kD monomer, 880-kD dimer), urease (272-kD trimer), and BSA (66-kD monomer, 132-kD dimer).…”

Characterization and Physiological Function of Class I Low-Molecular-Mass, Heat-Shock Protein Complex in Soybean

“…2,E and F). Nondenaturing gradient PAGE as the first dimension followed by SDSdenaturing PAGE as the second dimension (Clark and Critchley, 1992) revealed that this 280-kD LMW-HSP complex has a 15-to 18-kD protein as a major band, and other HMW HSPs, such as 82, 72, and 68 kD, with their native mass (from 150-115 kD) are minor components (Fig. 3B).…”

“…The LMW HSPs in vertebrates (Arrigo and Welch, 1987;Merck et al, 1993), Drosopkila (Arrigo and Pauli, 1988), yeasts (Bentley et al, 1992), and recently in plants (Clark and Critchley, 1992;Helm et al, 1993;Chen et al, 1994) were shown to form higher order structures similar to the native molecular mass of a-crystallin.…”