Knockdown of DAPIT (Diabetes-associated Protein in Insulin-sensitive Tissue) Results in Loss of ATP Synthase in Mitochondria

Shigenori Ohsakaya,Makoto Fujikawa,Toru Hisabori,Masasuke Yoshida

doi:10.1074/jbc.m110.198523

Abstract

It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α- and β-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.

Highlights

Cc.kyoto-su.ac.jp. 3 The abbreviations used are: DAPIT, diabetes-associated protein in insulin-sensitive tissue; CCCP, carbonyl cyanide m-chlorophenylhydrazone; TMRE, tetramethylrhodamine ethylester; voltage-dependent anion channel protein (VDAC), voltage-dependent anion channel
DAPIT-knockdown Cells Lose Population of ATP Synthase in Mitochondria—mitochondrial fractions of shDAPIT cells were analyzed with Clear Native PAGE, and ␣-subunit, ␤-subunit, and DAPIT were visualized by Western blotting (Fig. 2A)
DAPIT is not essential for function and structure of mature ATP synthase. It is lost from ATP synthase during purification [12] or by exposure to relatively strong detergent [13] without losing the structural integrity and the core function of ATP synthase [13]

Summary

EXPERIMENTAL PROCEDURES

Cells and Materials—HeLa and Phoenix GP cells were purchased from Health Science Research Resources Bank (JCRB9004) and American Type Culture Collection (SD-3514), respectively. They were cultured at 37 °C in a 5% CO2 incubator in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical) supplemented with 10% FBS (Invitrogen). Antibodies against ATP5A (␣-subunit), ATP5B (␤-subunit), and voltage-dependent anion channel protein (VDAC) were purchased from Molecular Probes (A21350 and A21351) and Abcam (ab16816), respectively. HRP-linked anti-mouse and anti-rabbit antibodies were purchased from GE Healthcare (NA931 and NA934). Rabbit antibody against human DAPIT was obtained by using purified DAPIT with histidine tag at the C terminus expressed in Escherichia coli. Retroviral Gene Transduction—For knocking down of DAPIT, we used four shRNAs: shDAPIT-1 (TACCAGTTCACTGGTATTA), shDAPIT-2 (CTGTGTACTGGCCACATAT), shDAPIT-3 (GTTAAGGTCCAAAAAAACT), and shDAPIT-4

Loss of ATP Synthase in DAPIT Knockdown

RESULTS

DISCUSSION