S-Space College of Natural Sciences (자연과학대학) Dept. of Biological Sciences (생명과학부) Theses (Ph.D. / Sc.D._생명과학부)
진핵세포에서 translocon을 통한 막단백질의 삽입과정 연구
Translocon-mediated insertion of membrane proteins in eukaryotic cells
- Hyun Ah Kim
- 자연과학대학 생명과학부
- Issue Date
- 서울대학교 대학원
- Membrane proteins; topology; GFP; glycosylation; ER; mitochondria; translocon; translocase.
- 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 2. 김현아.
- Membrane proteins contribute up to 30% of the proteome and are major targets for therapeutics. They transduce signals and transport macromolecules across the membrane. The structural information is lacking for most membrane proteins, despite that it is required to understand their function and to design appropriate drugs that target them, due to the difficulties in crystallization and a limited number of experimental tools.
The topology (2-dimensional structure) of membrane proteins are predicted by numerous bioinformatics programs, but it must be validated in vivo. However, the eukaryotic system lacks the topology reporter that allows live-cell assessment. In this thesis, glycosylatable GFP (gGFP) was developed as a novel topology reporter to deduce the topology of membrane proteins in yeast (Saccharomyces cerevisiae) and mammalian cells. gGFP was made by introducing a sequon (N-linked glycosylation site, N-X-T/S) near the fluorophore. gGFP was non-glycosylated and fluorescent in the cytosol, but became glycosylated and non-fluorescent in the ER lumen. Hence, the fluorescence and the glycosylation status provide the direct evidence of the localization of gGFP, allowing a rapid screening of membrane protein topology.
Membrane proteins adopt the correct topology during the biogenesis. They are inserted into the lipid bilayer through translocon complexes. The key subunits of translocon complexes are reported, however, how a translocon recognizes a transmembrane segment (TMS), mediates membrane insertion and determine final topology is not fully understood. The thesis aimed to understand the mechanism of translocon mediated membrane protein insertion at the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM).
The SEC61 complex mediates membrane protein insertion at the ER. When a TMS enters the translocon, Sec61 opens laterally towards the lipid bilayer at the interface between TMS2 and 7 (lateral gating helices) and allows the partitioning of a TMS into the membrane. Previous mutational analysis on Sec61 suggested that the insertion process is not a pure thermodynamic event, rather Sec61 is actively involved in the insertion process. To provide further insight into the opening and closing of Sec61 via the lateral gating helices, a systematic mutagenesis on TMS2 and 7 was performed in yeast. In the study, two groups of residues that either favor the open or closed conformation were identified. Compared to yeast SEC61, mammalian SEC61 complex contains additional subunits. To extend the investigation to mammalian system by characterizing the roles of different subunits of mammalian SEC61, the same set of model signal anchor proteins and multi-spanning membrane proteins used in the yeast study were expressed in HEK-293T and HeLa cells.
Unlike the SEC61 complex where the lateral gating helices of Sec61 regulate membrane protein insertion to the ER, how the TIM23 complex mediates membrane protein insertion into the mitochondrial IM remain elusive. Mgr2, a subunit of TIM23 complex, was termed as a lateral gate keeper as its expression level directly affected the insertion of Cyb2-DHFR and Mgm1. To test whether Mgr2 acts as a general gate keeper of the TIM23 complex and sets the hydrophobicity requirement for protein insertion, model mitochondrial IM proteins were expressed. Neither the insertion of other mitochondrial IM proteins nor the hydrophobicity requirement was altered at different Mgr2 expression levels. Thus, Mgr2s role in the gate keeping of TIM23 may be specific for Cyb2-DHFR and Mgm1.
Some mitochondrial IM proteins are inserted into the membrane directly by the TIM23 (stop-transfer pathway), whereas the others are sorted to the matrix first and inserted from the matrix side (conservative sorting pathway). Detailed bioinformatics analysis revealed that conservative sorting proteins tend to carry a proline residue in the TMS. To investigate the molecular mechanism of TMS recognition by Tim23, with a particular interest in how it discriminates a TMS with a proline, a screening scheme was designed and validated for the selection of Tim23 with an enhanced tolerance for a proline residue.