Structural stabilities of different regions of the titin I27 domain contribute differently to unfolding upon mitochondrial protein import.
Protein import into mitochondria requires unfolding of the folded mature domain of precursor proteins. Here we compared the effects of amino-acid replacement between the core region and the N-terminal region of the titin I27 domain (the 27th Ig domain of human titin) on its import into isolated mitochondria when attached to a short presequence (pb(2)(35)). We found that several mutations in the core region around Trp34 of the I27 domain enhanced the import rates of the fusion proteins, while the N-terminal K6P mutation, which increases mechanical stability around the N-terminal region, decreases the import rate. When the K6P mutation is combined with core-destabilizing mutations, the import rates of the fusion proteins still decrease, unless a long segment is deleted. These results suggest that mutations in the core region could destabilize the transition state for unfolding from the intermediate with the detached N-terminal segment during import, leading to enhanced unfolding rates, although stabilization of the N-terminal region masks these effects. In other words, the rate-limiting step of the global unfolding upon import into mitochondria switches, depending on the balance between the stability of the N-terminal structure and the stability of the core region of the I27 domain.
Oguro
Takaomi
T
Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Yagawa
Keisuke
K
Momose
Takaki
T
Sato
Takehiro
T
Yamano
Koji
K
Endo
Toshiya
T
D002942:Circular Dichroism; D064211:Connectin; D006801:Humans; D018625:Microscopy, Atomic Force; D008928:Mitochondria / Q000378:metabolism*; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013050:Spectrometry, Fluorescence
D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases