The products of the tuberous sclerosis complex (TSC) genes, hamartin and tuberin, form a heterodimer. Recently we reported that hamartin directly interacted with Hsp70. However, the physiological implications of this interaction have not yet been clearly defined. Here we show that hamartin localized to the outer membrane of the mitochondria in an Hsp70-dependent manner. Moreover, phosphorylation of the T417 residue of hamartin was required for its localization to the mitochondria as well as its interaction with Hsp70. A non-phosphorylatable hamartin mutant at residue T417 was unable to localize to the mitochondria and suppress apoptosis, whereas non-phosphorylatable hamartin mutants T357A and T390A localized to the mitochondria and suppressed apoptosis. Importantly, non-phosphorylatable mutants (T357A, T390A and T417A) promoted apoptosis after treatment with Hsp 70-inhibitor KNK437. We conclude that hamartin inhibited apoptosis by localizing to the mitochondria and that its phosphorylation and binding to Hsp70 was required for facilitation of this process. (C) 2009 Elsevier Inc. All rights reserved.