The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.

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Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 mol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera ⌬ 1 -pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steadystate levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.The high levels of free Pro observed in some plant tissues and organs suggest that this amino acid may have an important function in normal plant growth and development. Very high levels of free Pro have been reported in the flowers and seeds of Arabidopsis (Chiang and Dandekar, 1995;Savouré et al., 1995), in inflorescences and siliques of Brassica napus (Flasinski and Rogozinska, 1985), in ovules of broad bean (Venekamp and Koot, 1984), in pollen grains of petunia and tomato (Zhang et al., 1982;Fujita et al., 1998), and in the mature fruits of citrus species (Clements and Leland, 1962), pear species (Ulrich and Thaler, 1955), and grapevine (Vitis vinifera; Lafon-Lafourcade and Guimberteau, 1962;Kliewer, 1968;Ough and Stashak, 1974). The role of free Pro in the development or function of these organs remains unknown. Similarly, the regulation and temporal patterns of Pro biosynthesis and accumulation during normal plant development, in the absence of abiotic stress, remain essentially uncharacterized.Most of the research concerning Pro metabolism in plants has been focused on its accumulation in vegetative tissues in response to abiotic stresses such as drought and salinity. Stress-induced accumulation occurs predominantly through the enhanced biosynthesis of Pro from Glu via the pathway catalyzed by P5CS and P5CR rather than from Orn via the pathway catalyzed by OAT and P5CR (Boggess et al., 1976;Rhodes and Bressan, 1986;Delauney and Verma, 1993). The onset of stress-induced Pro accumulation is correlated with transcriptional activation of the gene encoding P5CS, which is the key regulatory and ratelimiting enzyme in this biosynthetic pathway (Hu et al., 1992;Delauney and Verma, 1993;Kishor et al., 1995;Savouré et al., 1995;Yoshiba et al., 1995;Zhang et al., 1995;Peng et al., 1996;Strizhov et al., 1997). Transcriptional regulation of the gene encoding PDH, the firs...

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein (ϳ21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic (ϳ40 kDa), microsomal (ϳ50 kDa), and additional mitochondrial (ϳ22 kDa, mtGrpE#2) GrpE-like members. While a cytosolic GrpElike protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed ϳ47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3-untranslated region of the mtGrpE#2 transcript which are not present in the mtGrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different posttranscriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

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