Helliwell, Stephen, Filipuzzi, Ireos, Steffen, Janos and Koehler, Carla (2017) Stendomycin selectively inhibits TIM23-dependent mitochondrial protein import. Nature Chemical Biology. ISSN 1552-44501552-4469

Abstract

Stendomycin is an antifungal lipopeptide isolated from Streptomyces endus. In mammalian cells in culture (HeLa) Stendomycin triggers mitophagy causing significant and rapid loss of the mitochondrial marker Tom20. Counter screening suggests that this mitophagy is triggered by the uncoupling of mitochondrial membrane potential. Interestingly, we show that in S. cerevisiae Stendomycin inhibits Tim17 / Tim23-dependent, but not Tim22-dependent, protein import into mitochondria via the inner membrane translocon. Only TIM17 and TIM23 heterozygotes are significantly sensitive to Stendomycin in S. cerevisiae haploinsufficiency profiling. Stendomycin causes a rapid loss of normal mitochondrial morphology visualized with GFP-Cox4 or mitotracker. Selection of mutants resistant to Stendomycin in a growth assay reveals two point mutations in Tim17, Tim17G20D and Tim17L122W. These mutations increase significantly the IC50 of stendomycin from 0.8 uM to 2.4 uM (L122W) or 2.6 uM (G20S). The mitochondrial morphological effects of Stendomycin are abrogated in cells carrying Tim17G20D. In vitro assessment of protein import into intact purified mitochondria revealed that at a dose that had no effect on membrane potential the import of Tim17/Tim23 cargo, but not Tim22 cargo, was inhibited. The Tim17G20D mutant significantly abrogated the effects of Stendomycin on Tim17/Tim23-dependent import. In similar in vitro conditions membrane potential modulation was observed with doses 1000x higher than those required for complete Tim17/Tim23 import blockage. This effect on membrane potential is independent of the Tim17 compound resistant mutants. Thus Stendomycin has a dual action, first as a specific Tim17 / Tim23 inhibitor, and second as an mitochondrial membrane potential uncoupler apparently independent of Tim17 / Tim23.

Item Type:	Article
Date Deposited:	22 Nov 2017 00:45
Last Modified:	22 Nov 2017 00:45
URI:	https://oak.novartis.com/id/eprint/23818