Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The protein transports from the cell cytosol to the mitochondria are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. (1) In the TOM complex, Tom70 functions as the receptor for mitochondria precursors with internal targeting signals. In our Tom70 crystal structure, the C-terminal domain of Tom70 forms a large pocket which may represent the binding site for mitochondrial precursor and the N-terminal domain of Tom70 may function to gate the pocket. Interestingly, the gating of the precursor-binding pocket of Tom70 is regulated by Hsp70/Hsp90 binding. The crystal structure of Tom70-Hsp70/Hsp90 complex indicates that the C-terminal EEVD motifs of Hsp70/Hsp90 can maintain Tom70 in the open conformation for receiving mitochondrial precursor. To fully understand the mechanism how Tom70 interacts with its peptide substrate under the regulation of Hsp70/Hsp90, we have identified a peptide substrate P70-8 for Tom70-Hsp70 complex by phage display library screening. The crystal structure of Tom70-Hsp70 EEVD motif-peptide substrate complex will illustrate the mechanism how Tom70 functions as a receptor for the molecular chaperone-bound mitochondrial precursor in the TOM translocon. Structure-based mutagenesis studies will be performed to confirm our hypothesis. (2) In the TIM23 translocon, Tim50 functions as a receptor to guide the precursor with the N-terminal presequence to the inner membrane protein channel Tim23 for translocation. Tim50IMS may interact with the presequence. Tim50IMS can also interact with Tim23IMS to deliver the precursors to the transmembrane channel formed by the C-terminal domain of Tim23. Our crystal structure of Tim50IMS indicated a protruding ?-hairpin may represent the binding site for Tim23. Close to this ?-hairpin, Tim50 contains a large groove that may represent the binding site for the presequence. We intend to determine the crystal structures of Tim50IMS-presequence complex, Tim50IMS- Tim23IMS complex and Tim50IMS-Tim23IMS-presequence complex. (3) Tim23 represents the major component in TIM23 translocon and it forms the essential transmembrane channel in the mitochondrial inner membrane. To reveal the mechanism how this important membrane protein transports mitochondrial precursors, we propose to determine the crystal structure of Tim23. Tim23 has been known to be difficult to express using a number of systems. In preliminary data, we have developed a crystallization chaperone for yeast Tim23IMS using phage display library screening. We have successfully expressed Tim23 complexed with the crystallization chaperone using the """"""""self-cleaving"""""""" 2A peptide in Pichia system. The recombinant Tim23 is functional as shown by electrophysiological analysis using planar lipid bilayer system.

Public Health Relevance

Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The long-term goal of this proposal is to carry out structural and functional studies on TOM and TIM complexes to uncover the basic mechanisms how the translocons facilitate the protein translocations across the mitochondria outer and inner membranes. The proposed research may have broad impacts on human aging, cancer biology and diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM080261-05A1
Application #
8396716
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Ainsztein, Alexandra M
Project Start
2007-05-01
Project End
2016-04-30
Budget Start
2012-08-01
Budget End
2013-04-30
Support Year
5
Fiscal Year
2012
Total Cost
$293,000
Indirect Cost
$93,000
Name
University of Alabama Birmingham
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Wang, Peng; Li, Jingzhi; Sha, Bingdong (2016) The ER stress sensor PERK luminal domain functions as a molecular chaperone to interact with misfolded proteins. Acta Crystallogr D Struct Biol 72:1290-1297
Li, Jingzhi; Sha, Bingdong (2015) The structure of Tim50(164-361) suggests the mechanism by which Tim50 receives mitochondrial presequences. Acta Crystallogr F Struct Biol Commun 71:1146-51
Cui, Wenjun; Josyula, Ratnakar; Li, Jingzhi et al. (2011) Membrane binding mechanism of yeast mitochondrial peripheral membrane protein TIM44. Protein Pept Lett 18:718-25
Qian, Xinguo; Gebert, Michael; Hopker, Jan et al. (2011) Structural basis for the function of Tim50 in the mitochondrial presequence translocase. J Mol Biol 411:513-9
Cui, Wenjun; Li, Jingzhi; Ron, David et al. (2011) The structure of the PERK kinase domain suggests the mechanism for its activation. Acta Crystallogr D Biol Crystallogr 67:423-8
Tao, Jiahui; Petrova, Kseniya; Ron, David et al. (2010) Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR. J Mol Biol 397:1307-15
Li, Jingzhi; Cui, Wenjun; Sha, Bingdong (2010) The structural plasticity of Tom71 for mitochondrial precursor translocations. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:985-9
Li, Jingzhi; Qian, Xinguo; Hu, Junbin et al. (2009) Molecular chaperone Hsp70/Hsp90 prepares the mitochondrial outer membrane translocon receptor Tom71 for preprotein loading. J Biol Chem 284:23852-9
Hu, Junbin; Li, Jingzhi; Qian, Xinguo et al. (2009) The crystal structures of yeast Get3 suggest a mechanism for tail-anchored protein membrane insertion. PLoS One 4:e8061

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