the ygen The extracts fused by l Western were amino Cisd2, further of mitochondr
ID: 171548 • Letter: T
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The experimental evidence to show the Cisd2 localization in mitochondria are as follows:
1. They expressed EGFP- tagged ( Green Fluorescent protein) Cisd2 protein in NIH3T3 ( mouse embryonic fibroblast cells).
2. They found that Cisd2 was overlapping with the mitochondrial marker ( Evidence 1).
3. Also they found that the N-terminal 58 amino acids deletion lead to erosion of the mitochondrial localization. Then the N- terminal 58 amino acids were fused with EGFP which was then directed from nuclear and cytoplasmic localization to mitochondria thus confirming that the N-terminal 58 amino acids is necessary and sufficient to direct mitochondrial localization. (Evidence 2)
They also confimed the specific ( outer or inner membrane) localization of Cisd2 protein by the following process:
1. Seperation of mouse liver mitochondria into fractions of 'Outer membrane', 'Mitoplasts ( inner membrane and matrix)' and intermembrane space ( soluble material between inner membrane and outer membrane) was done. Immunoblotting each fraction revealed that Cisd2 was enriched in 'Outer membrane' fraction thus confirming that Cisd2 is a mitochondrial 'Outer membrane' protein. ( Figure 4C) ( Final Evidence)
Evidence 1: States to mean that Cisd2 is a mitochondrial protein.
Evidence 2: States to mean that 'N-terminal 58 amino acids' is necessary for proper localization of Cisd2.
Final evidence states to mean that since there was increased expression of Cisd2 in the outermembrane when the immunoblotting technique was done, thus confirms that Cisd2 is localized on outer membrane of mitochondria.
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