Structural components involved in plastid protein import

Import of preproteins into chloroplasts is an essential process, requiring two major multisubunit protein complexes that are embedded in the outer and inner chloroplast envelope membrane. Both the translocon of the outer chloroplast membrane (Toc), as well as the translocon of the inner chloroplast membrane (Tic) have been studied intensively with respect to their individual subunit compositions, functions and regulations. Recent advances in crystallography have increased our understanding of the operation of these proteins in terms of their interactions and regulation by conformational switching. Several subdomains of components of the Toc translocon have been studied at the structural level, among them the polypeptide transport-associated (POTRA) domain of the channel protein Toc75 and the GTPase domain of Toc34. In this review, we summarize and discuss the insight that has been gained from these structural analyses. In addition, we present the crystal structure of the Toc64 tetratrico-peptide repeat (TPR) domain in complex with the C-terminal domains of the heat-shock proteins (Hsp) Hsp90 and Hsp70.

Shading after anthesis in wheat influences the amount and relative composition of grain proteins

SUMMARYThe high molecular weight glutenin subunits (HMW-GS) and glutenin macropolymer (GMP) in wheat grain are important characteristics that affect the quality of wheat products. Light intensity, as one of the environmental factors affecting grain yield and quality, has been studied extensively; however, little is known about its impact on HMW-GS and distribution of GMP granules in wheat grain. In the present study, two strong-gluten winter wheat cultivars with different subunit compositions were used to evaluate the effect of shading at different grain-filling stages on changes in HMW-GS and distribution of GMP granules in wheat grains. No effects of shading on initial formation time of each individual subunit were found; they responded similarly to shading with an increase in relative content, though the accumulation amount per grain of each individual subunit was decreased due to a decrease in grain weight induced by shading. Shading at different grain-filling stages, especially at the middle grain-filling stage, led to a significant increase in GMP content during grain filling; however, the proportions (by volume, number and surface area) of the larger GMP granules were increased by shading at middle and late grain-filling stages and decreased by shading at early grain-filling stage. It was also found that the content of total HMW-GS was positively correlated with volume proportions of larger GMP granules and negatively correlated with volume proportions of small GMP granules, which indicated that the pattern of response of distribution of GMP granules to shading was closely related to the regulatory effect of shading on the HMW-GS.

Significance of arginine radicals for sturgeon gonadotropin secondary structure and function

Significance of arginine radicals for sturgeon gonadotropin secondary structure and function Guanidine groups of arginine side chains were selectively chemically modified with 1,2-cyclohexanedione (CHD) in sturgeon (Acipenser güldenstädti Br.) gonadotropic hormone (GTH) and in its subunits. It was found that only two of the six guanidines were accessible for the reagent and each of the two modified groups was bound to an individual subunit. The results showed that both modified groups were located on the surface of the hormone dimeric molecule. CD-spectroscopy of the modified hormonal preparations did not indicate any considerable changes in their secondary structure. On the basis of the data obtained, a conclusion was made that the free guanidine groups are of exclusive importance for the hormone function at the receptor level as the bearers of the positive charge in the functionally important active sites or effector zones located on the surface of the hormone molecule. Also, it was shown that the guanidine groups played a certain role in sustaining the functionally effective spatial structure of the subunits and GTH.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability ( Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.