TIM23 복합체를 통한 미토콘드리아 내막 단백질 삽입경로의 연구

학위논문 (석사)-- 서울대학교 대학원 : 생명과학부, 2014. 2. 김현아.Nucleus encoded mitochondrial inner membrane (MIM) proteins carrying a cleavable presequence in Saccharomyces cerevisiae are inserted into the inner membrane medicated by translocases of outer and inner membrane (TOM-TIM23 complex). A hydrophobic sorting signal within transmembrane (TM) domain of the protein is recognized and laterally transferred into the membrane from the TIM23 complex (stop-transfer pathway). Otherwise, entire protein is first imported into the matrix and exported into the inner membrane (conservative sorting pathway). In addition, a multi-spanning mitochondrial protein utilizes both pathways for its membrane integration. While mitochondrial inner membrane is the protein-richest membrane harboring various complexes for critical functions in the cell, only small number of MIM proteins insertion mechanisms have been characterized owing to lack of robust experimental tools. Here, we established Mgm1 fusion protein (MFP) approach, which takes advantage of the rhomboid cleavage region in the C-terminal domain of Mgm1p, to elucidate the sorting pathways of MIM proteins in vivo. We validated this method with a set of proteins whose sorting pathways are well characterized and determined the membrane insertion mode of single or multi-spanning MIM proteins of which integration pathways were unknown. Our results suggested that Yta10p is inserted into the membrane not from matrix, but from intermembrane space (IMS). In addition, we found that a subunit of succinate dehydrogenase, Sdh4p and a mitochondrial inner membrane half-type ABC transporter, Mdl2p, are also integrated into the membrane via two different pathways. Furthermore, analyzing the sorting pattern of Mgm1 fusion proteins in various growth conditions and at different yeast mutant strains, we showed that insertion of proteins bearing moderately hydrophobic TM segments is more sensitive to intrinsic and extrinsic cellular factors and the presequence translocase-associated import motor (PAM complex) is necessary for the translocation of Cox18p, Mdl1p, and Mdl2p.CONTENS List of Figures……………………………………………………iii List of Tables……………………………………………………iv Abstract……………………………………………………………v Introduction………………………………………………………1 Materials and Methods Yeast strains……………………………………………………5 Plasmid construction……………………………………………5 Western blot analysis……………………………………………5 Isolation of yeast mitochondria and protease K protection analysis……………………………………………………………6 [35S]-pulse labeling and chase experiment…………………7 Results Design of experiments……………………………………………8 MFPs inserted by the stop-transfer pathways mostly produce L-MFP…………………………………………………12 MFPs sorted by the conservative sorting pathways predominantly generate s-Mgm1p…………………………13 Yta10p is inserted into the membrane from the IMS………16 Mdl1p is sorted into the inner membrane by stop-transfer and conservative sorting pathways…………………………19 Insertion modes of MIM proteins with previously uncharacterized sorting mechanism are determined ……23 Growth under respiring conditions increases the import of MFPs carrying conservative sorting proteins into the matrix………………………………………………………………27 Growth at different temperatures shows varying membrane insertion efficiency for MFPs…………………………………27 Impairment of PAM16 function inhibits translocation and membrane insertion of Cox18p, Mdl1p and Mdl2p………30 Inactivation of m-AAA protease results in increment of membrane insertion……………………………………………34 Discussion………………………………………………………38 References………………………………………………………45 국문초록…………………………………………………………54Maste

Performance Behavior by H₂ and CO as a Fuel in Intermediate Temperature Solid Oxide Fuel Cell (IT-SOFC)

The performance behavior of solid oxide fuel cell using H₂ and CO as fuels was investigated. The power densities and impedance results showed a little variation as the ratio of H₂ and CO changed. However, when the pure CO was used as a fuel, area specific resistance (ASR), especially low frequency region, was increased. This might be due to carbon deposition on anode. The maximum power density was 60% lower using CO than using H₂. Carbon deposition reduced after constant current was applied. The SOFC performance was recovered from the carbon deposition after applying constant current during 100h

Study of Operation Strategy for Hybrid PEM Fuel Cell and Supercapacitor

PEMFC has several technical problems such as water management, long term stability and performance degradation as. PEMFC has been studied not only to solve water management, but also to generate power in stable manner to system by using a hybrid system with auxiliary energy storage device. The purpose of this study is to couple PEMFC with supercapacitor to make a hybrid system and to design and test control strategies for stable power generation in case of changing output power. The polarization curve and dynamic behaviors while changing power were investigated to find out characteristics of PEMFC stack. A DC/DC converter was fabricated in order to increase fuel cell and supercapacitor voltage and to charge supercapacitor. We found that the operation strategy 2 was recommended to the system because of solving water management problem and increasing the dynamic behavior