Hitoshi Inoue

Three Sets of Translocation Intermediates Are Formed during the Early Stage of Protein Import into Chloroplasts

During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far. In this study, an attempt was made to evaluate the extent of translocation of the precursor by determining the state of the precursor in the early intermediate under various conditions and analyzing the fragments generated by limited proteolysis of the precursors docked to chloroplasts. Our results indicate that three different sets of early intermediate are formed based on temperature and the hydrolysis of GTP/ATP. These have been identified based on the size of proteolytic fragments of the precursor as “energy-dependent association,” “insertion,” and “penetration” states. These findings suggest two individual ATP-hydrolyzing steps during the early stage of protein import, one of which is temperature-sensitive. Our results also demonstrate that translocation through the outer envelope membrane is mainly dependent on internal ATP.

Alternative Processing of Arabidopsis Hsp70 Precursors during Protein Import into Chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.

EVALUATING THE ENERGY-DEPENDENT BINDING IN THE EARLY STAGE OF PROTEIN IMPORT INTO CHLOROPLASTS

During protein import into chloroplasts, precursor proteins are synthesized in the cytosol with an amino-terminal extension signal and irreversibly bind to chloroplasts under stringent energy conditions, such as low levels of GTP/ATP and low temperature, to form the early translocation intermediates. Whether the states of the early-intermediates that are formed under different energy conditions are similar has not been well studied. To evaluate the early intermediate states, we analyzed how precursor proteins within the early intermediates behave by employing two different approaches, limited proteolysis and site-specific cross-linking. Our results indicate that three different combinations of three different early intermediate stages are present and that the extent of precursor translocation differs between these stages based upon temperature as well as hydrolysis of GTP and ATP. Furthermore, the transition from the second to the third stage was only observed by increasing the temperature. This transition is also accompanied by the hydrolysis of ATP and the movement of the transit peptide. These results suggest the presence of temperature-sensitive and temperature-insensitive ATP-hydrolyzing steps during the early stages of protein import.

The transition of early translocation intermediates in chloroplasts is accompanied by the movement of the targeting signal on the precursor protein.

During protein import into chloroplasts, precursor proteins are docked to these organelles under stringent energy conditions to form early translocation intermediates. Depending on the temperature and the requirement for ATP, different types of early-intermediates are present, for which the extent of precursor protein translocation differs [H. Inoue, M. Akita, J. Biol. Chem. 283 (2008) 7491-7502]. However, it has not been determined whether the environment surrounding the precursor differs for each intermediate. We therefore employed a site-specific photo-crosslinking strategy in our current study to capture any components in close proximity to the targeting signal of the precursors within the early-intermediates. Various crosslinked products, one of which contains Toc75, were identified. The appearance of these products was found to be dependent on the position of the precursor upon modification by the crosslinker and also the intermediate state. This indicated that the transition of early translocation intermediates is accompanied with the movement of the targeting signal within the early-intermediates.

Development and optimization of an in vitro chloroplastic protein import assay using recombinant proteins

The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization.

An improved technique for low anterior resection using a PDS endoloop

We describe herein the results of performing a new technique of low anterior resection of the rectum using a PDS endoloop, on ten patients with rectal cancer. This technique involves first preparing the rectosigmoid colon with an anvil as in the conventional low anterior resection; then, after the stapler is inserted transanally, two endoloops are slid over the colon and rectum. The rectum is ligated by pushing the knot of the endoloop and a second knot is applied 2 cm proximal to the first. Finally, the rectum is cut and the stapler is closed and fired to make a circular end-to-end anastomosis. The level of the anastomosis ranged from 2.5 to 6 cm with a mean of 4.7 cm in the ten patients, only one of whom developed a minor anastomotic leakage postoperatively. Moreover, no patient has developed local recurrence or distant metastasis to date. In summary, this technique offers certain advantages that allow the operation to be done with more skill and safety in a narrow pelvis.

Localized 18F-fluorodeoxyglucose uptake at the pancreatic head during remission phase of autoimmune pancreatitis: A case report

Autoimmune pancreatitis (AIP) is a unique form of pancreatitis, histopathologically characterized by dense lymphoplasmacytic infiltration and fibrosis of the pancreas with obliterative phlebitis. AIP is associated with a good response to steroid therapy. Differentiation between AIP and pancreatic cancer to determine a preoperative diagnosis is often challenging, despite the use of various diagnostic modalities, including computed tomography (CT), magnetic resonance imaging and endoscopic retrograde cholangiopancreatography. It has been reported that 18F-fluorodeoxyglucose (18F-FDG)-positron emission tomography (PET)/CT may be a useful tool for distinguishing between the two diseases. In the present case report, a 71-year-old male patient presented with a well-circumscribed, solitary, nodular and homogenous 18F-FDG uptake at the pancreatic head, while receiving maintenance steroid therapy in the remission phase of AIP; preoperatively, the patient had been strongly suspected of having pancreatic cancer. Pathological examination revealed post-treatment relapse of AIP. The present case highlights the diagnostic and management difficulties with AIP in the remission phase. In certain cases, it remains challenging to differentiate the two diseases, even using the latest modalities.

In vitro fluorescent analysis of preprotein import into chloroplasts.

Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541–549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.

A Case of Peutz-Jeghers Syndrome Associated with Advanced Jejunal Cancer.

我々は進行空腸癌を合併したPeutz-Jeghers症候群 (以下, P-J症) の1例を経験したので報告する. 症例は43歳, 男性のP-J症患者である. 腸閉塞症にて近医より当院を紹介された. 消化管精査の結果, 胃, 小腸および大腸に多発性のポリープを認め, 腸閉塞症状が反復するため外科的治療を行った. 開腹の結果, 腹膜播種を伴う全周性の空腸癌を認め, 組織学的診断は粘液癌であった. 本例は高度進行癌であったため, どのような経路を経て癌が発生したか推察できなかった. P-J症のポリープの癌化を示唆する報告が増えてきた現在, その癌化の機序は解明されるべき重要な課題であると考えられ, P-J症の診断確定時より癌の合併も念頭においた厳重な経過観察を行う必要があると思われた.