MCL-1 directly binds with Cisd2 and regulates mitochondrial metabolism Chih-Pin Chuu1, Jen-Chih Tseng1, Shih-Han Huang1, Ching-Yu Lin1, Bi-Juan Wang1, Ting-Fen Tsai2 1Institute of Cellular and System Medicine, National Health Research Institutes, Taiwan; 2Institute of Molecular and Genomic Medicine, National Health Research Institutes, Taiwan *Corresponding Author : Dr. Chih-Pin Chuu, Institute of Cellular and System Medicine, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 350, Taiwan. E-Mail: email@example.com.
The CISD2 gene is evolutionarily conserved and encodes a transmembrane protein associated with the mitochondrial outer membrane. Deficiency of Cisd2 has previously been shown to shorten lifespan, resulting in premature aging in mice. Cisd2 knockout causes nerve and muscle degeneration. Little is known for protein binding partners of Cisd2. We introduced Micro-Western Array (MWA) to perform high-throughput co-immunoprecipitation (CoIP) of CIsd2 protein. MWA is a modified reverse phase array composes of a GeSim Nanoplotter arrayer, a GE multiphor, and a Licor Odyssey infra-red scanner. MWA allows detecting protein expression level or phosphorylation status change of 96-384 different antibodies in 6-15 samples simultaneously. The result of Micro-Western Array is identical to conventional Western blotting assay. However, the quantity of samples and antibodies required for MWA is approximately 500-1000 fold less than conventional Western blotting. We performed high-throughput co-IP using MWA with 188 antibodies targeting important signaling pathways and identified 27 proteins interacting with Cisd2 proteins. Among these proteins, we are extremely interested in the myeloid cell leukemia factor 1 (Mcl-1). Mcl-1, a Bcl-2 family protein, exists in two forms as the result of alternative splicing. The long variant (Mcl-1L) acts as an antiapoptotic factor, whereas the short isoform (Mcl-1S) displays proapoptotic activity. The balance between the long and short variants of the Mcl-1 gene represents a key aspect of the regulation of mitochondrial physiology. In this study, our immunofluorescent microscopy (IF) indicated that Mcl-1 co-localizes with Cisd2. Split YFP assay suggested the possibility that Mcl-1 directly binds with Cisd2. Bcl-2 has been reported to interact directly with Cisd2 via BH3 and BH4 domains. We therefore generated different truncated forms of Mcl-1 with different combination of deletion of BH1, BH2, BH3, or BH4. Co-IP experiment indicated that BH4 is essential for the interaction between MCl-1 and Cisd2. Knockdown of Cisd2 increased oxygen consumption rate (OCR) of mitochondria. Knockdown of MCl-1 blocked the effect of Cisd2 knockdown on mitochondria OCR. In summary, we demonstrated that Mcl-1 directly binds with Cisd2 and interaction between Mcl-1 and Cisd2 may play important on regulation of mitochondrial metabolism.
Funding: This study was supported by CS-105-PP-14 (National Health Research Institutes) and MOST 105-2314-B-400-020, MOST 105-2628-B-400-005-MY3, MOST 105-2923-B-400-001-MY3 (Ministry of Science and Technology) for CPC in Taiwan.
Credits: None available.
You must be logged in and own this product in order to post comments.