# Activator of apoptosis harakiri (HRK) localisation at mitochondria alters mitochondrial morphology independently of other BCL‐2 proteins

## TL;DR

This study reveals that HRK, a protein involved in cell death, changes mitochondrial shape in a way that doesn't rely on its usual death-inducing function.

## Contribution

The study identifies a new role for HRK in altering mitochondrial morphology independent of its BH3 domain and apoptosis function.

## Key findings

- HRK induces apoptosis via its BH3 domain but not its transmembrane domain.
- HRK causes mitochondrial aggregation without affecting cristae structure or respiration.
- Mitochondrial reorganisation by HRK requires the transmembrane domain and upstream amino acids.

## Abstract

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.

How the structure of HRK, a member of the BCL‐2 protein family, contributes to its function is poorly understood. Functional analysis of HRK mutants reveals that HRK induces apoptosis in a BH3‐domain‐dependent, but transmembrane‐domain‐independent, manner. The transmembrane domain localises HRK to mitochondria, but alone is insufficient. These observations uncover a previously unknown role of HRK in modulating mitochondrial morphology that is independent of its BH3 domain and pro‐death function.