The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The biogenesis of chloroplasts is genetically complex, involving hundreds of genes distributed between the nucleus and organelle. In higher plants, developmental parameters confer an added layer of complexity upon the genetic control of chloroplast biogenesis: the properties of plastids differ dramatically between different cell types. While the biochemistry and structure of different plastid types have been described in detail, factors that determine the timing and localization of chloroplast development and that mediate chloroplast assembly have remained elusive. To identify nuclear genes that play novel roles in chloroplast biogenesis, we are exploiting nuclear mutations that block the accumulation of subsets of chloroplast proteins. Detailed study of the mutant phenotypes provides clues concerning the primary defect in each mutant. Mutants with defects in chloroplast translation and mRNA metabolism have been identified. Other mutants defective in the accumulation of multiple thylakoid complexes show no apparent defect in the synthesis of the missing proteins. These may identify factors involved in the integration of proteins into the thylakoid membrane and their assembly into functional complexes.

Light excitation of as little as 0.05% of the rhodopsin in a retinal rod membrane suspension reduces the near-IR optical transmission by 25%. This transmission decrease requires the presence of guanosine triphosphate, is opposite in sign and 25 times larger in amplitude than a GTPdependent light-scattering signal previously reported in rod outer segment suspensions [Kuhn, H., Bennett, N., MichelVallez, M. & Chabre, M. (1981) Proc. Natl. Acad. Sci. USA, 78,[6873][6874][6875][6876][6877], and is kinetically complex. The initial phase of the optical transmission decrease begins after about a 50-ms lag (at 0.05% bleach) and has a first-order time constant of 300-500 ms. The scattering signal returns to the preactinic baseline in a time dependent on the amount of GTP added. A nonhydrolyzable GTP analogue, guanylyl imidodiphosphate, produces a scattering signal that does not return to the preactinic baseline. Adenosine triphosphate strongly inhibits the return of the GTP-dependent transmission decrease to the preactinic baseline. This effect of ATP on the GTP signal apparently requires ATP hydrolysis because it is inhibited by the simultaneous presence of adenylyl imidodiphosphate, a nonhydrolyzable analogue of ATP. The light-scattering signal and the velocity of the activation of a rod outer segment phosphodiesterase saturate when >0.05% of the rhodopsin is bleached and both show nearly identical dependence on light stimulus. It is suggested that these nucleotide-dependent light-scattering signals arise from changes in the state of membrane aggregation that are controlled by enzymatic processes. This hypothesis is supported by the large amplitude of the signals, sedimentation experiments, and a strong membrane concentration dependence. The ATP effects can be rationalized within the above hypothesis as being due to ATP-dependent rhodopsin phosphorylation that adds negative charges to the membrane surface and tends to keep the membranes disaggregated. An additional signal, which increases light transmission, is produced by a second, much more intense flash. The latter signal is interpreted as the result of proton binding by bleached rhodopsin molecules that decreases the negative charge repulsion between the membranes and allows increased aggregation.Retinal rod outer segments (ROS) contain several proteins, other than the visual pigment rhodopsin, whose activities are modulated by light (1)(2)(3)(4). The fact that these proteins are light activated makes it likely that they participate in the process of visual transduction or light adaptation. It is clear that an active form of bleached rhodopsin, R*, catalyzes the binding of GTP to a GTP-binding protein (3, 5-7). Once GTP is bound, the binding protein then activates a phosphodiesterase (PDE) until the bound GTP is hydrolyzed (1,8). R* catalyzes the binding of GTP to a large number of GTP-binding protein molecules until it is inactivated by either slow thermal processes or faster phosphorylation by rhodopsin kinase (ref. 9; unpublished data). Kuhn et al. (10...

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A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced ≥40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-Sed function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.