Structural And Functional Studies Of The Yeast Mitochondrial Tim23 Channel And Mba1 Receptor

This thesis consist of two chapters.Chapter one:The molecular mechanism of the mitochondrial inner membrane Tim23 channel protein transport precursor protein,Chapter two:The structural and functional studies of Mbal how to bind to ribosome and stabilize new peptide chain.Chapter one:Mitochondria contain more than 1000 proteins and 99%of them are encoded by the nuclear DNA.The precursor protein is translated and synthesized in the cytoplasmic ribosome.Tim23 is the core subunit of TIM23 translocase,a protein-conducting channel that recognizes and transfers the cytosol precursor protein into the matrix or provides a lateral gate for releasing it into the mitochondrial inner membrane(MIM).Yeast Tim23 contains 222 residues,Previous studies suggested that the N-terminal domain(approximately residues 1 to 100),a hydrophilic segment predicted to have coiled-coil motif and proposed to act as a receptor for the presequence of precursor proteins,exposes into the intermembrane space(IMS).Several previous studies demonstrated that purified Tim23 forms a protein-conducting channel that can be activated by membrane potential and in presence of a mimetic mitochondrial preprotein,a signaling peptide corresponding to the presequence of cytochrome c oxidase subunit ?(denoted as pCoxIV).While we have an emerging understanding of Tim23 translocation mechanism for the role that it plays under pathological conditions,The molecular mechanism of Tim23 importing precursor proteins is poorly understood due to the missing of three-dimensional(3D)structure.Specifically,How the precursor protein and membrane potential activate Tim23 channel is poorly understood.Here we present the solution structure of yeast Tim23 channel in complex with a mitochondrial presequence peptide in micelles as determined by high-resolution nuclear magnetic resonance(NMR),The structure of Tim23-Peptide complex is formed by two identical monomers and a completely disordered mitochondrial signaling peptide.The structure of Tim23-Peptide complex reveals novel features,For instance,a coiled-coil motif in the IMS interacts with the presequence peptide,Particularly,the conserved salt bridges together with the large side-chains of tryptophan and tyrosine locating at the membrane interface operate the gating of Tim23 channel.Furthermore,Hydrophilic residues in membrane are identified in forming the aqueous protein-conducting channel.Result from structure-based mutations electrophysiological analysis is consistent with the Tim23-Peptide structure.Overall,we presented and verified the solution structure of Tim23-Peptide complex and mechanistically addressed the key physiological properties of Tim23 channel,provided an unprecedent structural view into the molecular mechanism of presequence translocation.Chapter two:Yesat Mbal is a peripheral membrane protein that mainly located on the MIM.This protein is exposed into the matrix.As a receptor of the mitochondrial ribosome,On the one hand,Mbal can directly bind to the nascent peptide chain exit site of the ribosomal large subunit and play a important role in the process of transporting the nascent peptide chain into the MIM.On the other hand,Mbal also acts as a chaperone,protecting the nascent peptide chain.So far,the process by which the precursor protein encoded by mitochondrial DNA is transported into the MIM is poorly understood,and the 3D structure of the Mbal has not yet been resolved.The molecular mechanism of Mbal specifically bind to the ribosome and transport precursor protein into the MIM is unknown so far.We determine the 3D structure of Mbal by the high-resolution liquid-state NMR,at the same time we use Cryo-electron microscopy to achieve the 3D structure of the complex of Mbal-ribosome-nascent peptide,Compare and analyze the conformational change of Mbal in free state and binding state,thereby elucidating the molecular mechanism of Mbal how to bind to the receptor and transport the precursor protein into the MIM.At present,the solution structure of Mbal and the initial 9.8 angstroms low-resolution Mbal-ribosome-nascent peptide complex structure have been obtained,and subsequent experiments and structural refinement are still advancing.