Protein X is a secreted protein produced as a precursor form called pro-X. The maturation of Protein X requires that a short N-terminal segment be removed (propeptide cleaving). You have generated two antibodies that are called Anti-Pro and Anti-C. Anti-Pro binds to sequence in the N-terminal segment of pro-X, but only when pro-X is intact. Anti-C binds the C-terminal half of Protein X. You want to use these antibodies to determine where, within the vesicle trafficking pathway, propeptide cleavage occurs. You perform a cell fractionation procedure and obtain five test tubes containing different cellular compartments (Fractions 1-5). In addition to Anti-Pro and Anti-C, you also probe the fractions with antibodies to clathrin and COP-II proteins. Your results are summarized in the following Table. (2 points each, 6 points total) Reaction of fraction with specific antibody: Anti-Pro Anti-C Anti-COPII Anti-clathrin Fraction 1 Fraction 2 Fraction 3 Fraction 4 +- Fraction 5 (+ = reacts; +++ = strongly reacts; - = no detectable reaction; +/- = reacts weakly) + + +++ +++ + +++ +++ A. From this data, which fraction contains: cis-Golgi: plasma membrane: ER: trans-Golgi:
B. In which fraction (1-5) is the spring protein likely to be undergoing processing (cleavage) into its mature form and briefly explain WHY?
C. What does fraction 2 represent and WHY?