Accumulation, Stability, and Localization of a Major Chloroplast Heat-Shock Protein

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, >80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 + 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids. A organisms produce heat-shock proteins (HSPs) t in response to elevated temperatures and certain other stresses (Lindquist and Craig, 1988). Physiological and genetic data indicate that the production of HSPs during stress is essential for cell survival or recovery from stress. There are four major classes of HSPs in eukaryotes, HSP90, HSP70, HSP60 (or GroE), and low molecular weight (LMW) HSPs (15-30 kD). Proteins with homology to the first three classes are also synthesized by Escherichia coli in response to heat stress. Although HSPs were first identified by their elevated expression during stress, in many cells it has been found that HSPs or highly homologous proteins are expressed constitutively, or under cell cycle or developmental control (Lindquist and Craig, 1988). Furthermore, HSP60 and distinct homologues of HSP70 are present in semiautonomous organeiles, the mitochondria, and chloroplasts (Hemmingsen et al., 1988; Craig et al., 1989; Marshall et al., 1990). Chloroplasts also contain specific LMW HSPs (Kloppstech et al., 1985; Vierling et al., 1986). Clearly, HSPs are critical to multiple functions in eukaryotes. LMW, chloroplast-localized HSPs have been identified in diverse higher plant species, including both dicots and monocots (Kloppstech et al., 1985; Vierling et al., 1986, 1989). Each species produces a single, major LMW chloroplast HSP between 21 and 24 kD. These proteins are antigenically related and their corresponding cDNAs cross-hybridize. The proteins are nuclear encoded and are synthesized as 1. Abbreviations used in this paper: FNR, ferredoxin NADP reductase; HSP, heat-shock protein; LMW, low molecular weight. precursor polypeptides ~5 kD larger than the mature protein. The mRNA encoding the LMW chloroplast HSP has not been detected in unstressed leaf tissues, but it rapidly accumulates during heat stress to an estimated 0.75 % of the poly(A)RNA after 2 h at 38°C (Vierling et al., 1986). This is the most dramatic environmental regulation of any chloroplast protein studied to date. Using specific antibodies, the LMW chloroplast HSP was also shown to be undetectable in control tissues, although significant protein accumulated during stress (Vierling et al., 1989). The amino acid sequences of LMW chloroplast HSPs have been derived from the DNA sequence of cDNA clones from pea (Pisum sativum) (Vierling et al., 1988), soybean (Glycine max) (Vierling et al., 1988), Arabidopsis thaliana (Vierling, E., unpublished data), and petunia (Petunia hybrida) (Chen, Q., unpublished data). The carboxy-terminal domain of the mature protein ('~100 amino acids) is homologous to a domain found in LMW cytoplasmic HSPs and in the alphacrystallins, identifying these chloroplast HSPs as members of a eukaryotic superfamily of LMW HSPs (Lindquist and Craig, 1988; Vierling et al., 1988). The amino-terminal transit peptide which is removed during import into chloroplasts has characteristics typical of transit peptides from other nuclear-encoded chloroplast proteins (von Heijne et al., 1989). We have hypothesized that the chloroplast HSP arose from a duplication of a nuclear gene for a cytoplasmic protein which subsequently acquired amino-terminal sequences sufficient to direct it to the chloroplast (Vierling et al., 1988). © The Rockefeller University Press, 0021-9525/90/06/1873/11 $2.00 The Journal of Cell Biology, Volume 110, June 199