The enzyme dihydrofolate reductase (DHFR) normally resides in the cytosol, and it can be imported into mitochondria by appending a mitochondrial signal sequence. However, when this modified DHFR is incubated with methotrexate, which is a substrate analog that binds tightly to the active site, the modified DHFR is no longer imported. Propose an explanation for this finding. Required:
a. Consider a transmembrane protein that spans the inner nuclear membrane. Suggest a pathway by which such a protein could reach its destination.
b. Clathrin can assemble with multiple types of adaptors to create coated vesicles. Explain why this multiplicity of adaptors is useful to the cell

Within the mitochondria, the import takes place post-translationally. The imported protein must be in an unfolded condition so that it can go through the mitochondria's import machinery. In the existence of methotrexate, the bound DHFR moiety remains in the folded state, and thus, stays on the surface of mitochondria.

It is witnessed that the presequence must be of the length of 55 amino acids to cover both the membranes of mitochondria and must be of about sixty amino acids length to associate with the mhsp70. If the presequence of this methotrexate bound DHFR is of seventy-five amino acids in length, it can associate with mhsp70 within the matrix, to initiate the ATP reliant import within the mitochondria. Due to the presence of various endocytic and intracellular sorting signals, multiple adaptors are essential.

not only is it colder, it also becomes darker, less salty, and more dense.