「様式３－B」

課題番号（Project number） 12

Identification and characterization of mitochondrial
nucleoids［１］組織（Research group）
代表者（Principal Investigator (PI)）：
Lene Juel Rasmussen（University of Copenhagen）
対応者（Host researcher at IDAC）：
Akira Yasui, Shin-ichiro Kanno（IDAC, Tohoku University）
研究費 (Expenditure report of research funds)：
consumable goods 300,000 Yen，
［２］研究経過（Research setup）
In
the last decade it has become increasingly clearthat mitochondrial DNA (mitDNA) is not naked but
associated with proteins in structure called
nucleoids that are essential for mitDNA
maintenance. Mitochondrial transcription factor A(TFAM) is an essential component of mitochondrial
nucleoids and plays important roles inmitochondrial transcription and replication. The
purpose of the proposal is to identify andcharacterize proteins in mitochondrial nucleoids bydetermining TFAM complex.

by using domain-based affinity screening ofbinding proteins. All the experiments wereperformed in the division of dynamic proteome in
IDAC, Tohoku University. We had discussion
about the project and the results always withe-mail.
［３］成果（Research outcomes）（３－１）研究成果（Results）
For the analysis of TFAM function inmitochondria our approach is to identify theproteins from human mitochondrial extracts,
which interact with TFAM and its complex. We
applied a domain-based affinity screening ofbinding proteins developed in the IDAC
laboratory. There are five supposedly functional
domains in TFAM (Fig. 2A). We used the amino
half HMG domain indicated with N and theC-terminal half domain (C).
AFNCBGST

Fig. 1. The role of TFAM in mitochondria(Ventura-Clapier et al. Cardiovascular Research2008.)
Because of the importance of mtDNA
maintenance in cancer and aging we wanted toknow the structure of TFAM complex in details

F, N or C

Mitochondrial
extracts

Fig. 2. Experimental procedure
These regions were attached behind GST,expressed in E. coli and purified. Human
mitochondrial extracts were prepared from ahuman cell culture by conventional method in Dr.
V. Bohr’s laboratory as well as in IDAC.

Sample:*Mitochondria*ext.
GST$TFAM
GSTFN
CkDa
220
160
120
100

50

12
3430

12345678910111213222423
25
26
27

20

10

12345678*109111213141516171918
20215Fig. 3. Protein bands eluted after binding witheach GST-fusion proteins.
Five protein samples from the affinity withfull-length (F), 27 samples from N region and 13
samples from C region were determined by nano
LC-MS/MS analysis (Fig. 3).
While the details of the 45 interacting proteins
can not be disclosed in this report, the followingclassification of the TFAM-interacting proteins
was obtained;
1. The putative interactive proteins of TFAM
were divided into five categories; 1) mitochondria
metabolism 2) RNA metabolism, 3) translationand chaperon, other functions and 5) ribosomes.
2. We identified 5 proteins, of which function in
mitochondria have never been reported.
3. At least 12 proteins were identified, of whichinteraction with TFAM has never been reported.
4. Because of the interaction of TFAM withprotein modification enzymes, TFAM may be
involved in a process essential for protein
maintenance system within mitochondria.

（３－２）波及効果と発展性など（Futureperspectives）
This may be the first trial for the determination ofTFAM protein by a domain-based affinity
screening of binding proteins by using humanmitochondrial cell extracts. We obtained anumber of interesting candidate proteins ofTFAM interaction. These data suggest more
important roles of TFAM and the nucleoids
played in mitochondria than it is known. Basedon the results here obtained we will continue tofind out how mitochondrial genome integrity is
maintained and determine new roles of TFAM
complex.
［４］成果資料（List of Papers）
In preparation.