Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact
Takeshi Harada1, Ryota Sada1, Yoshito Osugi1
1Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan.
Abstract:
Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.
Insights
Cytoskeleton-associated protein 4 (CKAP4) is crucial for mitochondrial function. Its palmitoylation enables binding to VDAC2, maintaining mitochondrial calcium levels and energy production, impacting cancer cell growth.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Cancer Research
Background:
- Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated transmembrane protein.
- CKAP4 is localized to the endoplasmic reticulum (ER) and influences cellular structure.
- The role of CKAP4 in mitochondrial function and its interaction partners remain largely unexplored.
Purpose of the Study:
- To investigate the role of CKAP4 in mitochondrial structure and function.
- To identify proteins that bind to CKAP4.
- To elucidate the mechanism by which CKAP4 influences mitochondrial calcium and membrane potential.
Main Methods:
- Utilized CRISPR-Cas9 to generate CKAP4 knockout (KO) HeLaS3 cells.
- Performed co-immunoprecipitation to identify CKAP4 binding proteins.
- Assessed mitochondrial structure, ER-mitochondria contact sites, and VDAC2 binding.
- Measured intracellular calcium levels and mitochondrial membrane potential.
- Evaluated oxidative consumption rates, cancer cell proliferation, and tumor formation in vivo.
Main Results:
- CKAP4 KO altered mitochondrial morphology and increased ER-mitochondria contact sites.
- VDAC2 was identified as a CKAP4 binding partner, requiring CKAP4 palmitoylation at Cys100.
- CKAP4 KO enhanced IP3R-VDAC2 binding, increased mitochondrial Ca2+ levels, and decreased mitochondrial membrane potential.
- CKAP4 deficiency reduced oxidative consumption, low-glucose cancer cell proliferation, and xenograft tumor growth.
Conclusions:
- CKAP4 maintains mitochondrial function by binding to VDAC2 at ER-mitochondria contact sites.
- Palmitoylation of CKAP4 is essential for its interaction with VDAC2 and subsequent mitochondrial regulation.
- CKAP4 plays a significant role in cellular energy metabolism and cancer progression.
Related Concept Videos
01:19Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:39Mitochondrial Protein Sorting
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...
01:28The Inner Mitochondrial Membrane
01:45Mitochondrial Membranes
01:39Mitochondrial Precursor Proteins
Most of the mitochondrial...
01:12Porin Insertion in the Outer Mitochondrial Membrane
Three models describe the assembly of porins by the SAM complex and their insertion into the outer membrane. Model 1 suggests that porins are assembled outside the SAM channel as the...