Suppose you are interested in discovering new human proteins that are involved i

Question

Suppose you are interested in discovering new human proteins that are involved in trafficking other proteins from the cytoplasm to the mitochondria. However, your lab experience is in yeast. In precise detail, write an outline of an experimental design you would use for generating, verifying and isolating a haploid yeast strain with a mutation in a key protein in this specific trafficking process, and then describe how you would use this yeast strain to identify, isolate, and ultimately sequence a human cDNA that encodes a protein essential in this trafficking mechanism. For full credit, your outline must include all important steps.

Explanation / Answer

Most of the mitochondrial proteins are encoded by the nuclear genome, synthesised in the cytosol on free ribosomes as precursor proteins and then transported into or across mitochondrial membranes with the aid of a handful of distinct complexes. The transfer of mitochondrial proteins from their site of synthesis in the cytosol to the surface of the mitochondrial outer membrane, where translocation begins, mainly occurs in a post-translational manner that is, the fully synthesized protein contacts the mitochondrial import machinery. The proteins targeted to mitochondria from the cytoplasm will have a signal sequence by which they are destined to end up in the mitochondria.

Generating a mutant of new protein by deleting their signal sequences which will enable us to particularly to identify the function of these proteins in the trafficking of other proteins into mitochondria. Start by determining the effects of a complete knockout of the protein on cell function using a one-step gene replacement approach. To do this, you transform a PCR product harboring a selectable marker flanked by DNA sequences homologous to genomic regions flanking protein into wild-type diploid cells and plate the cells onto selective medium.

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