Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from the stroma has not been previously described. Genetics and localization studies were used to show that plastids contain two different Sec systems with distinct functions. Loss-of-function mutations in components of the previously described thylakoid-localized Sec system, designated as SCY1 (At2g18710), SECA1 (At4g01800), and SECE1 (At4g14870) in Arabidopsis (Arabidopsis thaliana), result in albino seedlings and sucrose-dependent heterotrophic growth. Loss-of-function mutations in components of the second Sec system, designated as SCY2 (At2g31530) and SECA2 (At1g21650) in Arabidopsis, result in arrest at the globular stage and embryo lethality. Promoter-swap experiments provided evidence that SCY1 and SCY2 are functionally nonredundant and perform different roles in the cell. Finally, chloroplast import and fractionation assays and immunogold localization of SCY2-green fluorescent protein fusion proteins in root tissues indicated that SCY2 is part of an envelope-localized Sec system. Our data suggest that SCY2 and SECA2 function in Sec-mediated integration and translocation processes at the inner envelope membrane.

SUMMARYThylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of and the gene encoding the fructose-specific phosphofructokinase () during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with and DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.

Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40.