Translocon-mediated insertion of membrane proteins in eukaryotic cells = 진핵세포에서 translocon을 통한 막단백질의 삽입과정 연구

다국어 초록 (Multilingual Abstract)

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, Mgr2’s 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.

목차 (Table of Contents)

• CHAPTER I - INTRODUCTION 1
• I.1. Membrane proteins and topology 2
• I.1.1. Types of membrane proteins 2
• I.1.2. Topology of membrane proteins 2
• I.1.3. Topology reporters 5
• I.2. Protein targeting and insertion into the ER membrane 6
• I.2.1. Co- and post-translational translocation pathway 6
• I.2.2. SEC61 translocon complex 6
• I.2.3. Determinants of ER membrane protein insertion 8
• I.3. Protein targeting and insertion into the mitochondrial IM 9
• I.3.1. Protein import pathways for mitochondrial proteins 9
• I.3.2. TIM23 complex 10
• I.3.3. Determinants of mitochondrial IM protein insertion 12
• I.4. Aims and experimental approaches 14
• I.4.1. Development of a glycosylatable GFP as a topology reporter 14
• I.4.2. Membrane protein insertion by SEC61 translocon of yeast and human cell-lines 15
• I.4.3. Assessment of Mgr2 in membrane protein insertion via TIM23 complex 18
• I.4.4. Screening for Tim23 with enhanced proline tolerance 20
• I.4.5. Quantitative analysis of protein import kinetics and forces 22
• CHAPTER II - MATERIALS AND METHODS 24
• II.1. Yeast and mammalian cell culture 25
• II.1.1. Glycosylatable GFP study 25
• II.1.2. Sec translocon study 25
• II.1.3. Mgr2 study 25
• II.1.4. Tim23 and optical tweezer study 26
• II.2. Proteasome inhibition assay 26
• II.3. Protein preparation, SDS-PAGE and Western blotting DNA manipulation 26
• II.4. Pulse (Chase) labeling, immuno-precipitation and autoradiography 27
• II.5. Optical tweezer based in vitro protein import assay 27
• II.5.1. Mitochondria isolation and in vitro protein import assay 27
• II.5.2. List of constructs made for in vitro protein import assay 27
• II.6. Fluorescence microscopy 28
• II.7. Fluorescence measurements 28
• CHAPTER III - RESULTS 29
• III.1. Glycosylatable GFP as a novel membrane protein topology reporter 30
• III.1.1. Engineering of glycosylatable GFP for Saccharomyces cerevisiae 30
• III.1.2. Validation of glycosylatable GFP as a topology reporter 35
• III.1.3. Development of glycosylatable GFP for mammalian cell culture 37
• III.1.4. Topology assessment of disease related proteins by glycosylatable GFP 43
• III.2. Structural profiling of the lateral gating helices of yeast Sec 46
• III.2.1. The residues lining TMS7 of Sec61 affects the targeting and insertion of a single-spanning membrane 46
• III.2.2. The residues lining TMS7 of Sec61 affects the insertion of a 2nd TMS of a double spanning membrane protein 49
• III.3. Profile of membrane protein insertion into mammalian ER 52
• III.3.1. Membrane insertion profile of single-spanning membrane proteins 52
• III.3.2. Membrane insertion profile of multi-spanning membrane proteins 55
• III.3.3. siRNA mediated silencing of Sec62 60
• III.3.4. Rescue of Sec62 knock-down 61
• III.3.5. Expression of human translocon subunits in yeast 61
• III.4. Lateral gating of Tim23 by Mgr2 is limited to Mgm1 and Cyb2 62
• III.4.1. Expression level of Mgr2 is low 62
• III.4.2. Mgr2 does not set the hydrophobicity requirement for membrane protein insertion into mitochondrial IM 64
• III.4.3. Mgr2 does not affect the sorting of mitochondrial IM proteins taking the stop-transfer or conservative sorting pathway 65
• III.4.4. Mgr2 senses the charges preceding the TMS of Mgm1 68
• III.4.5. Mitochondrial IM protein sorting in glycerol is unaffected by Mgr2 71
• III.4.6. Sorting kinetics of mitochondrial IM proteins 72
• III.5. TIM23 mutagenesis screening 73
• III.5.1. Validation of TIM23 mutagenesis selection scheme 73
• CHAPTER IV - DISCUSSION 75
• IV.1. Development of an ER topology reporter 76
• IV.1.1. Glycosylatable GFP as a novel ER topology reporter of eukaryotic cells 76
• IV.2. Protein insertion into the ER membrane by the SEC61 complex 76
• IV.2.1. The role of the lateral gating helices of Sec61 in membrane protein insertion 76
• IV.2.2. A platform to study the SEC61 complex mediated membrane protein insertion in mammalian cell-lines 77
• IV.3. Protein insertion into the mitochondrial IM by the TIM23 complex 78
• IV.3.1. Mgr2 may not act as a general gate keeper of the TIM23 complex 78
• IV.3.2. The molecular mechanism of the TIM23 complex mediated protein import and insertion into the membrane 79
• REFERENCES 80
• 국문초록 90