<p dir="ltr">Premature aging disorders such as mandibuloacral dysplasia disorder (MAD) can be caused by improper maturation of nuclear scaffolding protein lamin A from its precursor prelamin A. ZMPSTE24 is responsible for both the earlier C-terminal CAAX cleavage and a subsequent N-terminal upstream cleavage during the posttranslational processing of prelamin A to lamin A. Although ZMPSTE24’s structure and function are well characterized, the role of the 4 apparent openings into its hollow inner chamber remains unknown. We hypothesize that one of these entrances, portal 1, is the initial substrate entry point based on its proximity to the zinc-coordinating active site. Unfortunately, ZMPSTE24 is difficult to express and purify. Fortunately, the yeast homolog, Ste24, not only shares many structural and functional similarities to ZMPSTE24 but is also much easier to express and purify in an active state. Therefore, we will use Ste24 and its substrate <b>a</b>-factor as a model system for ZMPSTE24 and its substrate prelamin A to deduce whether portal 1 acts as the primary substrate entry point. We examined portal 1’s function in primary substrate entry by observing how the incubation of portal 1 mutants engineered with cysteine residues around the portal with cysteine-reactive bismaleimide crosslinkers affects the activity of the C-terminal CAAX cleavage. If crosslinking of the cysteine residues occludes the portal, we hypothesize that activity will decrease because substrate cannot enter. The cysteine-less enzyme Ste24 (QA), which cannot react with the crosslinkers, was engineered by mutagenesis to contain 1 or 2 new cysteines at specific positions around this portal. We hypothesize that portal 1 occlusion with cysteine reactive bismaleimide crosslinkers will inactivate the enzyme by preventing substrate entry. We monitored changes in CAAX cleavage activity with a radioactive endoprotease-coupled CAAX assay.</p><p dir="ltr">In crude membranes derived from yeast expressing QA Ste24, activity was not inhibited in the presence of either BMH or BMOE crosslinker. For the single cysteine-containing mutants M210C, T267C, I307C, and V311C, each crosslinker similarly decreased activity over 50%. For the double cysteine-containing mutants M210C-I307C, T267C-I307C, and T267C-V311C, we found between 20-60% decreased activity in the presence of the crosslinker which has a length most similar to the distance between the two cysteines. These results closely reflect previous data and further suggest that CAAX activity of the enzyme may be decreased due to the occlusion of the primary entry site, portal 1.</p><p dir="ltr">With purified QA Ste24, changes in activity were less apparent. Activity for purified QA was not decreased in the presence of either crosslinker. Single cysteine-containing mutants did not show decreased activity in the presence of either crosslinker. Unlike what was observed in crude membrane preparations, the double-cysteine containing mutants exhibited minimal decrease in activity in the presence of the crosslinker that has a length most similar to the distance between the two cysteines.</p><p dir="ltr">In crude membrane preparations, the cysteine-containing QA Ste24 mutants have diminished activity in the presence of crosslinkers. This may be due to the occlusion of the primary entry point, portal 1. However, we recognize the possibility that the decrease in activity was the result of the occlusion of the exit portal site. It is imperative that further experiments confirm that exit portal occlusion is not occurring. For purified cysteine-containing QA Ste24 mutants, the negligible decrease in activity suggests either that the portal 1 is not the primary substrate entry point or that the conditions of the assay were not optimized to generate inhibition. For example, the concentration of crosslinker was not sufficient in the presence of excess lipid and the reconstitution mixture sequestered the crosslinker. Further optimization of the reaction conditions is warranted. The cysteine-containing QA Ste24 mutants must be assessed for free thiols to determine how successful the reaction with the crosslinkers is. A more developed understanding of how all four portals function in the Ste24 CAAX processing of <b>a</b>-factor will be very insightful towards the mechanism of ZMPSTE24 in lamin A CAAX processing and may catalyze new targets of study for ZMPSTE24-related diseases like MAD-B.</p>