Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondr ial anion carrier proteins (MATP). Localized in the inner membrane of the o rganelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis a nd, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, th e yeast two-hybrid system was applied. Segments of hUCP2 containing the hyd rophilic loops facing the intermembrane space, or combinations of these, we re used to screen an adipocyte activation domain (AD) fusion library. The 1 4.3.3 protein isoforms theta, beta, zeta were identified as possible intera cting partners of hUCP2. Screening of a human skeletal muscle AD fusion lib rary, on the other hand, yielded several clones all of them encoding the ga mma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal interm embrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between U CP3 and 14.3.3 theta could be demonstrated after in vitro translation by co immunoprecipitation. When coexpressed in a heterologous yeast system, 14.3. 3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in t he targeting of UCPs to the mitochondria.