The Evolutionary Conserved Transmembrane BAX Inhibitor Motif (TMBIM) Containing Protein Family Members 5 and 6 Are Essential for the Development and Survival of Drosophila melanogaster

Abstract

The mammalian Transmembrane BAX Inhibitor Motif (TMBIM) protein family consists of six evolutionarily conserved hydrophobic proteins that affect programmed cell death and the regulation of intracellular calcium levels. The bacterial ortholog BsYetJ is a pH-dependent calcium channel. We here identified seven TMBIM family members in Drosophila melanogaster and describe their expression levels in diverse tissues and developmental stages. A phylogenetic analysis revealed that CG30379 represents the ortholog of human TMBIM4 although these two proteins are much less related than TMBIM5 (CG2076 and CG1287/Mics1) and TMBIM6 (CG7188/Bi-1) to their respective orthologs. For TMBIM1-3 the assignment is more dubious because the fly and the human proteins cluster together. We conducted a functional analysis based on expression levels and the availability of RNAi lines. This revealed that the ubiquitous knockdown of CG3798/Nmda1 and CG3814/Lfg had no effect on development while knockdown of CG2076/dTmbim5 resulted in death at the pupa stage and knockdown of CG7188/dTmbim6 in death at the embryonic stage. Ubiquitous knockdown of the second TMBIM5 paralog CG1287/Mics1 ensued in male sterility. Knockdown of dTmbim5 and 6 in muscle and neural tissue also greatly reduced lifespan through different mechanisms. Knockdown of the mitochondrial family member dTmbim5 resulted in reduced ATP production and a pro-apoptotic expression profile while knockdown of the ER protein dTmbim6 increased the ER calcium levels similar to findings in mammalian cells. Our data demonstrate that dTmbim5 and 6 are essential for fly development and survival but affect cell survival through different mechanisms.

TMBIM5 loss of function alters mitochondrial matrix ion homeostasis and causes a skeletal myopathy

“…Absence of TMBIM5 protein was confirmed by immunoblotting ( Fig 2A ). TMBIM5 -deficient cells have a disrupted cristae architecture with loss of cristae ( Fig 2B ) and reduced maximal uncoupled mitochondrial respiration as shown by a Seahorse analysis ( Fig 2C ) similar to previously reported TMBIM5 loss-of-function cellular phenotypes ( Oka et al, 2008 ; Seitaj et al, 2020 ; Zhang et al, 2021 ). Based on these findings, we concluded that our TMBIM5 KO HEK293 cells constitute a bona fide model to study the effects of TMBIM5 loss of function on mitochondrial Ca 2+ dynamics.…”

“…To study the endogenous function of TMBIM5, we generated TMBIM5-deficient HEK293 cells using CRISPR/Cas9. Absence of TMBIM5 protein was confirmed by immunoblotting (Fig 2A (Oka et al, 2008;Seitaj et al, 2020;Zhang et al, 2021). Based on these findings, we concluded that our TMBIM5 KO HEK293 cells constitute a bona fide model to study the effects of TMBIM5 loss of function on mitochondrial Ca 2+ dynamics.…”

“…Both proteins are evolutionarily conserved. Ubiquitous knockdown of dmTmbim5 in Drosophila melanogaster results in lethality at the pupa stage of development ( Zhang et al, 2021 ). We hypothesized that TMBIM5 might be implicated in mitochondrial ion homeostasis because it is the mitochondrial member of a family of six evolutionarily conserved hydrophobic TMBIM proteins, with the founding member being BAX inhibitor-1 (BI-1, also known as TMBIM6) ( Lisak et al, 2015 ).…”