A cDNA Encoding the Endoplasmic Reticulum-Luminal Heat-Shock Protein from Spinach (Spinacia oleracea L.)

HSP70s are an evolutionarily conserved family of 70-kD proteins better known as molecular chaperones (Ellis and van der Vies, 1991). Molecular chaperones assist the in vivo assembly and folding of polypeptides during normal growth without themselves becoming part of the final folded protein (Gatenby, 1992). HSP70s show differential expression to a variety of abiotic or biotic factors (for review, see Craig, 1989; Gething and Sambrook, 1992). In eukaryotes, one of the best-characterized organellar HSP70s is that of the ER-luminal HSC70. During normal growth the ER-luminal HSC70 is required for translocation, folding, and assembly of secretory and transmembrane proteins passing through the ER secretory pathway (Vogel et al., 1990). However, the ER-luminal HSC70s will bind to misfolded, underglycosylated, and mutant polypeptides, which in tum generally causes the increased expression of the HSC70 (Hendershot et al., 1988; Fontes et al., 1991). Like other HSP70s, the ER-luminal HSC70 contains a highly conserved N-terminal ATP-binding domain and shows increased ATP hydrolysis (Flynn et al., 1989) when peptides are bound at the less-conserved C-terminal peptide-binding domain. The ER-luminal HSC70 also undergoes autophosphorylation of a Thr residue (Freiden et al., 1992), which may have a regulatory role in its function. A cDNA library made from cold-acclimated spinach (Spinacia oleracea L.) leaf tissue in XZapII was screened with polyclonal antibody to spinach HSC70s (Neven et al., 1992) and with polymerase chain reaction-generated spin350 (Neven et al., 1992). This led to the isolation of a partia1 clone (p73) of 1387 bp, which was identified as a C-terminal portion of the ER-luminal HSC70. Clone p73 was then used to further screen a cDNA library for full-length clones of the ER-luminal HSC70. p522 was the largest clone isolated (2436 bp). Sequence homology analysis showed that the amino acid sequence from p522 has a 91.5% homology to the ER-luminal HSC70 (BLP4) of tobacco (Denecke et al., 1991). Southem blots indicated that the spinach ER-luminal HSC70 is encoded by a single gene (our unpublished data), which contrasts with that reported for tobacco (Denecke et al., 1991). The data in this report (Table I) show the characteristics of