longer than normal. This lack of life-span extension was particularly unexpected because mitochondrial respiration is widely assumed to influence aging in an ongoing manner during adulthood through the generation of reactive oxygen species (8, 20). Our findings bring this assumption into question.Caloric restriction during adulthood extends life-span and has been proposed to act by decreasing the rate of respiration (21, 22). However, our finding that life-span extension caused by respiratory-chain RNAi requires inhibition during development suggests that caloric restriction in animals, as in yeast (23), extends life-span in another way. The same holds for insulin/IGF-1 signaling, which functions exclusively during adulthood to influence C. elegans life-span (19).In conclusion, we propose that C. elegans possesses a regulatory system that senses, interprets, and remembers the rate of mitochondrial respiration during development. Under normal conditions, this system establishes normal rates of growth, behavior, and aging. However, if the rate of respiration is low, this system reduces the animal's growth rate and body size, as well as its rates of behavior and aging. It is possible that the rate of respiration during development is sufficient to specify the rate at which the animal lives its entire life; alternatively, the adult animal may make reference to contemporaneous rates of respiration, which, in turn, are influenced by mitochondrial activity during development. References and Notes Coordinated Nonvectorial Folding in a Newly Synthesized Multidomain ProteinAnnemieke Jansens,* Esther van Duijn, Ineke Braakman †The low-density lipoprotein receptor (LDL-R) is a typical example of a multidomain protein, for which in vivo folding is assumed to occur vectorially from the amino terminus to the carboxyl terminus. Using a pulse-chase approach in intact cells, we found instead that newly synthesized LDL-R molecules folded by way of "collapsed" intermediates that contained non-native disulfide bonds between distant cysteines. The most amino-terminal domain acquired its native conformation late in folding instead of during synthesis. Thus, productive LDL-R folding in a cell is not vectorial but is mostly posttranslational, and involves transient long-range nonnative disulfide bonds that are isomerized into native short-range cysteine pairs.

Previous work showed that from all cellular proteins, the endoplasmic reticulum (ER) resident proteins are most sensitive to oxidative stress [hydrogen peroxide (H(2)O(2))], as determined using the oxidation-sensitive, membrane-permeable, acetylTyrFluo probe. Because of the importance of these proteins in proper cellular functioning, we studied (a) whether modifying the cellular redox state/antioxidant status alters the susceptibility of those proteins toward H(2)O(2) oxidative stress and (b) whether H(2)O(2) affects ER function with regard to protein folding. The cellular redox and/or antioxidative capacity was modified in several ways. Lowering the capacity increased H(2)O(2)-induced protein oxidation, and increasing the capacity lowered H(2)O(2)-induced protein oxidation. The effect of H(2)O(2) on ER-related protein maturation was investigated, using the maturation of the low-density lipoprotein receptor as a model. Its maturation was not affected at low concentrations of H(2)O(2) (< or = 400 micro M), which do result in oxidation of ER resident proteins. Maturation was slowed down or reversibly inhibited at higher concentrations of H(2)O(2) (1.5-2.0 mM). These results might be caused by several events, including oxidation of the low-density lipoprotein receptor itself or ER resident proteins resulting in decreased folding (capacity). Alternatively, oxidation of cytosolic proteins involved in ER Golgi transport might attenuate transport and maturation. Clearly, the mechanism(s) responsible for the impairment of maturation need further investigation.

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulsechase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.

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