Activator of apoptosis harakiri ( <scp>HRK</scp> ) localisation at mitochondria alters mitochondrial morphology independently of other <scp>BCL</scp> ‐2 proteins

The activator of apoptosis harakiri (HRK) is a pro-apoptotic BCL-2 homology 3 (BH3)-only protein of the apoptosis regulator Bcl-2 (BCL-2) family that is mainly expressed in neuronal and haematopoietic tissues. How specific HRK protein domains contribute to its pro-apoptotic function, and what other non-apoptotic roles HRK performs within cells, remain poorly understood. Here, we evaluated the apoptosis sensitivity, and mitochondrial shape and function of HCT116 human colorectal cells lacking all BH3-only proteins as well as all relevant BCL-2 proteins. By reconstituting individual BH3-only proteins on this genetic background, we observed that HRK induces apoptosis in a manner dependent on its BH3 domain, and the presence of the apoptosis regulator BAX and BCL-2 homologous antagonist/killer (BAK), but independent of its transmembrane domain. Intriguingly, HRK also causes mitochondrial aggregation without altering cristae structure or respiration. Although the BH3 domain is not required for mitochondrial reorganisation, we found that the transmembrane domain requires additional upstream amino acids for HRK mitochondrial localisation and reorganisation. These observations uncover a previously unknown role of HRK in modulating mitochondrial morphology that is independent of its BH3 domain and pro-death function.