Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death

Drob-1, a Drosophila member of the Bcl-2/CED-9 family that promotes cell death

^*Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, ^‡Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), and ^§Strategic Promotion System for Brain Science (SPSBS), Science and Technology Agency of Japan at Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan; and ^†Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109

Edited by H. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA, and approved November 23, 1999 (received for review September 30, 1999)

Abstract

The Bcl-2/CED-9 family of proteins, which includes both antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report here the identification and characterization of Drob-1, the first Drosophila member of the Bcl-2/CED-9 family to be isolated. Drob-1 contains four conserved Bcl-2 homology domains (BH1, BH2, BH3, and BH4) and a C-terminal hydrophobic domain. Ectopic expression of Drob-1 in the developing Drosophila eye resulted in a rough-eye phenotype. Furthermore, when overexpressed in Drosophila S2 cells, Drob-1 induced apoptosis accompanied by elevated caspase activity. This Drob-1-induced cell death, however, could not be antagonized by baculovirus p35, a broad-spectrum caspase inhibitor. Drob-1 was localized to the intracytoplasmic membranes, predominantly to the mitochondrial membranes, and a mutant Drob-1 lacking the hydrophobic C terminus lost both its mitochondrial localization and its proapoptotic activity. These results suggest that Drob-1 promotes cell death by inducing both caspase-dependent and -independent pathways at the mitochondria. Our identification of Drob-1 and further genetic analysis should provide increased understanding of the universal mechanisms by which the Bcl-2/CED-9 family members and other related proteins regulate apoptosis.

Footnotes

↵ ¶ To whom reprint requests should be addressed at: Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, 2–2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: mmiura{at}nana.med.osaka-u.ac.jp.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AB032430).
Abbreviations:

BH,

Bcl-2 homology;

DIAP,

Drosophila inhibitor-of-apoptosis protein;

GMR,

glass multimer reporter;

UAS,

upstream activation sequence;

GFP,

green fluorescent protein;

CED,

cell death abnormal;

RT-PCR,

reverse transcription–PCR;

HA,

hemagglutinin;

HM,

heavy membrane;

LM,

light membrane