2015 Fiscal Year Annual Research Report
| Project/Area Number |
15J05033
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| Research Institution | Nagoya University |
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Principal Investigator |
宋 紀瑶 名古屋大学, 理学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2015-04-24 – 2017-03-31
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| Keywords | Tom40 channel / yeast / mitochondria |
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| Outline of Annual Research Achievements |
We reported that yeast Om45, one of the major proteins of the yeast mitochondrial outer membrane (OM), is anchored to the OM from the intermembrane space (IMS) and is transported to the IMS by a novel import pathway involving the membrane potential across the inner membrane and the TIM23 complex (Song et al., EMBO Rep., 2014). However, we found that when C-terminally fused to DHFR, topology of the Om45 fusion protein flips. A tightly folded C-terminal domain appears to block the transport by the Om45 pathway, so that the protein could be laterally released into the OM with the Nin-Cout topology. Therefore, I would like to test the possibility of lateral release of OM proteins from the Tom40 channel. To this end, I established threes types of fusion proteins that block the Om45 pathway: I, the Om45-pathway targeting signal fused to a loosely folded domain followed by DHFR; II, the Om45-pathway targeting signal directly fused to PINK1; III, the Om45-pathway targeting signal directly fused to DHFR. For fusion proteins type I and II, I observed intermediate formation at the TOM40 Complex level. I assume that these proteins are stuck at the Tom40 channel or stay close to it. For the Type III substrates, I did not observe any intermediate. I assume that this kind of fusion proteins are laterally released from the Tom40 channel into the OM quickly and then leave the TOM40 Complex. Therefore, the type III fusion proteins may be the most suitable for investigating the possibility of lateral release of proteins from the Tom40 channel.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
To test the possibility of lateral release of OM proteins from the Tom40 channel. To this end, I need proper substrate proteins. To eliminate the situation that Om45N40-DHFR can fall off the Tom40 channel and then insert back into the OM spontaneously, I fused N-terminal 220 amino acids to of cytochrome b2 to the N-terminus of Om45N30-DHFR or DHFR. In this case, the heme-binding domain of b2 will be folded in the inter-membrane space (IMS) therefore preventing the fusion proteins from slipping off the Tom40 channel. Next, I used the site-specific photocrosslinking method to analyze interactions of the fusion protein with the Tom40 channel (Masatoshi Esaki et al., nsb1008, 2003). Theoretically, if there is lateral gating of Tom40, b2 fusion protein contains a transmembrane domain (TMD) could be laterally released into the OM. Indeed, I observed less cross linking products from b2(220)-OM45N30-DHFR with Tom40 compare with that of b2(220)-DHFR.
To the same end, I adopted the non-stop protein approach. Proteins without a stop codon will be stalled on ribosomes and cannot be released, and over express of nonstop proteins can cause stuck of their import channel. Previously, we observed growth defects of yeast expressing Leu9 non-stop (Toshiaki Izawa et al., Cell Reports 2, 2012). Thus, I fused the TMD of Om45 or Tom70 to the C-terminus of Leu9 non-stop and observed growth recover in dom34 and ski7 deletion background. This suggests that Leu9-Om45N or Leu9-Tom70N nonstop has left the Tom40 channel.
From the results I've gotten, it is promising to prove the lateral gating of Tom40.
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| Strategy for Future Research Activity |
Next, i will construct different length of cytochrome b2 fuse with the TMD of Om45 or Tom70 follow by DHFR. Then I will perform blue native PAGE after import of RI-labeled fusion proteins with or without a TMD into Tom40-FLAG mitochondria, to compare the interaction of those fusion proteins with the Tom40 channel. If fusion protein with a TMD cannot form intermediate with Tom40, it suggests the lateral gating of the Tom40 channel. In the meanwhile, I will use newly constructed fusion proteins for site-specific photocrosslinking to get constant results. To prove the assumption that Tom40 channel is lateral opening farther more, I will test the effect of the Tom40 channel closure (Jian Qiu et al., Cell 154, 2013) on possible lateral release of the fusion proteins.
On the other hand, for in vivo non-stop protein approach, to eliminate the possibility that the non-stop fusion protein gets recognized as abnormal existence and be degraded by quality control. I will use dom34, ski7and Ltn1 triple deletion strain as background. Since stuck of the Tom40 channel can cause accumulation of precursor form of matrix-targeted proteins. Next I will check the precursor form of Hsp60 in triple deletion yeast expressing Leu9, Leu9-Om45N or Leu9-Tom70N non-stop. For testing the interaction of the Leu9 non-stop proteins with and without a TMD, I will perform co-immunoprecipatation. To ensure that Leu9-Om45N or Leu9-Tom70N nonstop is indeed inserted into the OM, I will use alkaline extraction for assessing the membrane inserted form.
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Research Products
(3 results)
