4. Your friend is studying a secreted protein called Inp1. Inp1 is produced as a

Question

4. Your friend is studying a secreted protein called Inp1. Inp1 is produced as a precursor form called pro-Inp1. The maturation of Inp1 requires that a short N-terminal segment be cleaved. Your friend has raised two sets of antibodies that he calls Pro and C-Inp1. Pro antibodies specifically bind to the peptide removed by cleavage during maturation. C-Inp1 antibodies were raised using the C-terminal half of Inp1 as the antigen. Your friend wanted to use these antibodies to determine where within the secretory pathway propeptide cleavage occurs. He collected three different cellular fractions that contain ER, cis Golgi, and trans Golgi. He was about to assay these fractions with his Pro antibody and his Inp1 antibody when he realized that the labels had come off his cellular fractions in the freezer. He comes to you for sympathy, but you tell him to stop whining, run the fractions, and also probe the fractions with anti-COPII and anti-clathrin antibodies you have in your lab. He comes to you with his results, which are summarized in Table 1. From these results, can you determine in which compartment Inp1 is likely to be processed into the mature form? Explain.

Explanation / Answer

The protein Inp1 is most likely processed in the trans-Golgi compartment. That is because vesicles carrying cargo pinch off from the trans-Golgi and these vesicles are composed of clathrin. Since the protein would be processed here, the N terminus (Pro antibody) will show very less intensity in the Western blotting as it is getting cleaved. However, it shows very good reactivity in other fractions which also show good reactivity to COPII vesicles. COPII vesicles pinch off from the ER towards cis-Golgi and both the Pro and Inp1 (C terminus) antibodies show good reactivity in both these fractions. Hence, it is trans-Golgi where the protein is processed.

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