Long Li pawg ntawm Tsev Kawm Ntawv ntawm Kev Tshawb Fawb Txog Lub Neej, Peking University tau luam tawm ib daim ntawv hu ua "Molecular pathway of mitochondrial preprotein import through the Molecular pathway of mitochondrial preprotein import through the TOM-TIM23 supercomplex", luam tawm hauv Nature Structural & Molecular Biology. Txoj kev tshawb no qhia txog cov txheej txheem thiab biochemical cov txiaj ntsig ntawm TOM-TIM23 supercomplex uas kho cov protein thauj hauv mitochondria, nthuav tawm txoj hauv kev tshiab ntawm cov protein ntshuam los ntawm TOM complex hla lub mitochondrial txheej membrane, thiab redefines cov ntsiab lus tseem ceeb ntawm TIM23 transporter complex nyob rau hauv lub puab. mitochondrial membrane.
Raws li ib tug ntawm cov tseem ceeb tshaj plaws organelles nyob rau hauv eukaryotic hlwb, mitochondria plays lub luag hauj lwm tseem ceeb nyob rau hauv lub zog mov, intracellular metabolism, homeostasis thiab apoptosis. Mitochondria muaj ib qho tshwj xeeb sab hauv thiab sab nrauv bilayer lipid membrane qauv, nrog ntau tshaj 1, 000 cov proteins faib rau hauv. 99% ntawm mitochondrial proteins yog encoded los ntawm cytosolic genes, synthesized nyob rau hauv ribosomes nyob rau hauv lub cytoplasm, thiab thauj hla daim nyias nyias mus rau lub mitochondria ua lawv txoj haujlwm. Yog li ntawd, kev thauj mus los ntawm mitochondrial proteins yog qhov tseem ceeb rau kev tswj hwm thiab tswj kev ua haujlwm ntawm mitochondrial, thiab kev hloov pauv hauv cov noob caj noob ces zoo sib xws nrog kev loj hlob ntawm ntau cov kab mob metabolic thiab mob qog noj ntshav hauv tib neeg. Li Long cov pab pawg tau ua haujlwm los tshawb txog cov txheej txheem molecular uas cov mitochondrial proteins nkag mus rau mitochondria, thiab cov haujlwm tshaj tawm suav nrog kev daws txoj hauv kev uas cov mitochondrial membrane cov protein nkag mus rau hauv daim nyias nyias ntawm TIM22 thauj complex (Zhang Y. et al Cell Research 2021). Lub luag haujlwm ntawm TOM-TIM23 supercomplex hauv mitochondrial protein thauj yog qhov tseem ceeb tshaj hauv txoj kev tshawb no. Qhov no supercomplex, sib sau ua ke los ntawm kaum ob ntawm cov protein subunits, spans lub puab thiab sab mitochondrial bilayer daim nyias nyias thiab tswj kev thauj ntawm ntau tshaj 500 mitochondrial soluble thiab membrane proteins. Tshaj li 40 xyoo dhau los, cov kws tshawb fawb tau nthuav dav thiab siv zog kawm txog tus kheej subunits ntawm TOM thiab TIM23 thauj complexes. Txawm li cas los xij, vim qhov muaj zog heev ntawm qhov supercomplex no, kev nkag siab ntawm lub hauv paus lo lus nug ntawm yuav ua li cas cov subunits sib sau ua ke los tsim txoj hauv kev thauj cov protein tseem txwv, tshwj xeeb tshaj yog rau TIM23 complex nyob rau hauv lub endosomal membrane, qhov twg muaj pes tsawg leeg ntawm cov tub ntxhais. tsheb thauj mus los tau tsis meej heev.
Hauv txoj kev tshawb no, cov kws sau ntawv tau pib ua ntej nrog kev sib sau ua ke ntawm TOM-TIM23 supercomplex ruaj khov, tshawb txog ntau yam kev sib dhos, thiab thaum kawg xaiv fuse ntsuab fluorescent protein nrog mitochondrial protein ua lub thauj substrate, thiab ntes lub xeev nruab nrab ntawm TOM-TIM23. supercomplex rau thauj cov protein substrates hauv cov poov xab. Tom qab nyob rau hauv vitro purification, lub xeev nruab nrab no tseem tuaj yeem ruaj khov rau kev tshawb fawb txog biology thiab biochemistry ntxiv. Los ntawm ib qho kev tsom xam ntawm qhov nruab nrab ntawm lub xeev no los ntawm cryo-electron microscopy, cov kws sau ntawv pom tias muaj kev cuam tshuam ncaj qha ntawm TOM complex nyob rau hauv daim nyias nyias thiab TIM23 complex nyob rau hauv daim nyias nyias, txuas lub puab thiab sab mitochondrial membranes. , uas zoo xa cov protein substrate los ntawm cov txheej txheem mitochondrial mus rau hauv daim nyias nyias (Fig. 1a). Tshwj xeeb, TOM complex tau daws qhov kev daws teeb meem ntawm 4.1 Å. Cov qauv qhia "polar amino acid txoj hauv kev" nyob rau hauv lub puab phab ntsa ntawm Tom40 channel subunit, uas yog khaws cia nyob rau hauv ntau hom thiab kho kom haum rau txoj kev ntawm cov protein substrate los ntawm lub Tom40 intermediate pore nyob rau hauv ib tug unfolded daim ntawv (Fig. 1b). Txoj kev no txawv ntawm ob txoj hauv kev transmembrane hauv Tom40 yav dhau los tau npaj tseg raws li kev sim tshuaj biochemical, ua kom pom qhov ua tau zoo ntawm TOM complex hauv kev paub zoo thiab thauj ntau yam mitochondrial proteins.
Daim duab 1. Cov qauv ntawm TOM-TIM23 supercomplex. (a) Schematic sib dhos thiab cryo-electron microscopy qauv ntawm TOM-TIM23 supercomplex. (b) Cryo-electron microscopy qauv qauv ntawm TOM complex uas muaj cov protein substrate. (c) AlphaFold2 tus qauv qauv ntawm Tim17-Tim23-Mgr2 heterotrimer
Tsis tas li ntawd, cov kws sau ntawv tau qhia txog qhov tsis zoo ntawm cov amino acids ntawm cov chaw sib txawv ntawm cov protein substrate thiab tshawb xyuas qhov chaw ib puag ncig ntawm cov protein substrate hauv txoj kev thauj mus los ntawm TOM-TIM23 los ntawm kev siv photocrosslinking, yog li txheeb xyuas cov tub ntxhais subunit uas ncaj qha pab lub substrate hla lub ob lipid daim nyias nyias. Cov txiaj ntsig kev sim tau pom tias C-terminal fragment ntawm cov protein substrate tuaj yeem sib txuas nrog Tom40 subunit ntawm daim nyias nyias, qhov tshwm sim zoo ib yam nrog cov qauv kev soj ntsuam. Kuj ceeb tias, txawm li cas los xij, N-terminal fragment ntawm cov protein substrate cross-links nrog Tim17 thiab Mgr2 subunits nyob rau hauv daim nyias nyias, tab sis tsis nrog Tim23 subunit, uas yog feem ntau pom tias yog tsim los ntawm lub puab membrane thauj channel. Qhov kev tshwm sim no qhia tias tej zaum yuav muaj qhov tsis txaus ntseeg tseem ceeb hauv kev nkag siab ntawm txoj hauv kev thauj mus los ntawm TIM23 complex hauv kev tshawb fawb yav dhau los. Txhawm rau txhawm rau txheeb xyuas cov tub ntxhais thauj khoom thauj mus los thiab txoj hauv kev hauv TIM23, cov kws sau ntawv siv kev sib xyaw ntawm AlphaFold2 qauv, biochemical cross-linking, poov xab noob, thiab hauv vitro thauj cov mitochondrial proteins, thiab pom tias cov ntsiab lus tseem ceeb ntawm TIM23 complex yog heterotrimer. suav nrog Tim23, Tim17, thiab Mgr2 subunits, nrog Tim17 thiab Mgr2 lub ntsej muag-rau-lub ntsej muag tsim cov qauv zoo li channel, thaum Tim23 khi rau Tim17 hauv daim ntawv rov qab thiab tsis koom nrog kev tsim channel (Daim duab 1c ). Thaum lub sij hawm hloov pauv cov protein, cov protein substrate hla dhau los ntawm cov qauv channel zoo li tsim los ntawm Tim17 thiab Mgr2 thiab tsis ncaj qha hu rau Tim23 subunit. Mutagenesis thwmsim qhia tias knockdown ntawm Mgr2 hauv cov poov xab tsis cuam tshuam rau kev hloov pauv ntawm cov protein, qhia tias tsuas yog Tim17 yuav tsum muaj nyob rau hauv cov qauv zoo li cov channel tsim los ntawm Tim17 thiab Mgr2.Qhov laj thawj vim li cas Tim17 tuaj yeem pab cov protein kom hla lub endosomal membrane yog vim nws qhov tshwj xeeb. nto amino acid faib: ntawm ib sab tes, Tim17 muaj ib tug heev conserved tsis zoo nqi cheeb tsam ntawm lub nkag ntawm lub endosomal membrane uas yuav cuam tshuam lub zos nyob rau hauv ib tes, Tim17 muaj ib tug heev conserved tsis zoo nqi cheeb tsam nyob rau hauv lub nkag ntawm lub puab daim nyias nyias. , uas tuaj yeem cuam tshuam cov qauv hauv zos phospholipid bilayer thiab txo lub zog thaiv rau cov protein kom hla cov lipid membrane; Ntawm qhov tod tes, Tim17 muaj thaj tsam hydrophobic khaws cia ntawm qhov chaw nruab nrab ntawm txoj kev, uas yuav pab ua kom cov ntaub so ntswg sab hauv ntawm mitochondrion nyob rau hauv ib qho chaw airtight thiab tswj cov membrane muaj peev xwm tsim nyog rau mitochondrial physiological kev ua ub no. Ua ke cov txiaj ntsig ntawm ntau yam qauv thiab kev sim tshuaj biochemical, nws tuaj yeem txiav txim siab tias TIM23 complex, nrog Tim17 subunit ntawm nws cov tub ntxhais, siv hom sib xyaw los pab cov protein hloov pauv, piv txwv li, Tim17 tuaj yeem cuam tshuam nrog lwm cov subunit los tsim ib qho channel los pab cov proteins thoob plaws hauv daim nyias nyias, los yog Tim17 ua hauj lwm nyob rau hauv lub insertion enzyme ua hauj lwm hom kom ua tiav cov protein translocation ib leeg yam tsis tas yuav tsum tau rau cov channel tsim. Qhov txiaj ntsig no kho qhov kev nkag siab tsis zoo hauv thaj chaw dhau 40 xyoo dhau los uas Tim23 subunit suav nrog cov tub ntxhais channel.
Hauv cov ntsiab lus, txoj kev tshawb no tau tsim ib lub tswv yim tshiab rau cov kev tshawb fawb thauj cov protein, qhia txog txoj hauv kev tseem ceeb rau mitochondrial proteins kom hla lub bilayer lipid membrane ntawm TOM-TIM23 supercomplex (Daim duab 2), overturned qhov ntev-sawv teb kev paub ntawm cov ntsiab lus tseem ceeb ntawm lub puab membrane TIM23 thauj complex, thiab nrhiav tau ib tug kiag li tshiab hom ntawm cov protein thauj enzyme ua hauj lwm, uas nteg ib tug ruaj lub hauv paus rau ib tug dav thiab in-tog nkag siab ntawm mitochondrial biosynthesis. synthesis, tso lub hauv paus ruaj khov rau kev nkag siab zoo thiab tob tob ntawm mitochondrial biosynthesis.
Daim duab 2. mitochondrial TOM-TIM23 transporter pathway
Long Li yog tus sau cov ntaub ntawv sib xws, thiab Xueyin Zhou, PhD cov tub ntxhais kawm los ntawm Peking University-Tsinghua Joint Center for Life Sciences chav kawm xyoo 2018, Yuqi Yang, PhD cov tub ntxhais kawm los ntawm Lub Tsev Kawm Ntawv ntawm Lub Neej Sciences chav kawm xyoo 2019, Dr. Peng Wang los ntawm Peking University cryo-electron microscopy platform, thiab Shanshan Wang, PhD cov tub ntxhais kawm los ntawm Lub Tsev Kawm Ntawv ntawm Kev Tshawb Fawb Txog Lub Neej Xyoo 2020, yog thawj tus kws sau ntawv. Dongjie Sun, yav dhau los tus kws tshaj lij hauv Li Long's lab, Xiaomin Ou, PhD cov tub ntxhais kawm hauv College of Life Sciences, chav kawm xyoo 2022, thiab Yuke Lian, PhD cov tub ntxhais kawm hauv College of Life Sciences, chav kawm xyoo 2021, tau ua haujlwm tseem ceeb rau qhov kev tshawb fawb no. Prof. Ning Gao thiab Associate Researcher Ningning Li los ntawm Lub Tsev Kawm Ntawv ntawm Lub Neej Sciences, Peking University, thiab Tus Kws Tshawb Fawb Xinzheng Zhang los ntawm Lub Tsev Haujlwm ntawm Biophysics, Suav Academy ntawm Kev Tshawb Fawb, tau muab kev pabcuam zoo hauv cryo-electron microscopy data xam, thiab Prof. Qing Li thiab Prof. Tau txais Chang los ntawm Lub Tsev Kawm Ntawv ntawm Kev Tshawb Fawb Txog Lub Neej, Peking University, tau muab cov lus qhia tseem ceeb ntawm cov noob caj noob ces thiab kev sim photocrosslinking, raws li. Cov haujlwm tshawb fawb tau txhawb nqa los ntawm Peking University cryo-electron microscopy platform, High Performance Computing Center, Instrumentation Center of School of Life Sciences, thiab National Phoenix Protein Platform, thiab tau txais nyiaj los ntawm State Key Laboratory of Membrane Biology, Peking University-Tsinghua Joint Center rau Life Sciences, thiab National Natural Science Foundation ntawm Tuam Tshoj.
