The glycogenolysosome: A protein interaction network determined by students in an undergraduate hypothesis‐driven cell biology laboratory course

At Western Kentucky University, we use a research‐oriented approach to teach the cell biology laboratory (Biol 412). The course is divided into two projects organized in an independent learning and discovery environment. One of these projects is a 10‐week long yeast two‐hybrid screen. Over the past three years, students have screened two proteins known to regulate glycogenolysis, glycogen phosphorylase (GPb) and phosporylase kinase (PhK), against a rabbit skeletal muscle cDNA library to search for protein‐protein interactions. From these experiments, students have identified several novel associations. Specifically, using two different constructs of GPb as bait, the regulatory domain (a.a. 17‐484) or the catalytic domain (a.a. 485–843), students identified muscle creatine kinase (M‐CK) and phosphoglycerate mutase as binding partners. As a validation for this screen, one student group, using the regulatory domain as bait, identified a positive association with GPb that corresponded to the C‐terminus of this protein. When the β subunit of PhK was used as bait, a positive association was identified also with M‐CK, as well as skeletal muscle α‐actinin, actin, and the α subunit of PhK (residues 424–579). The identification of PhK α was again a good positive control for the experiment. The identification of M‐CK as a binding partner with both PhK and GPb suggests that the CK pathway may serve as the primary source of ATP production for these enzymes during periods of high energy demand. Thus, by using various baits over successive years in the Biol 412 course, we have assembled a protein interaction network which we termed the “glycogenolysosome”. Moreover, this course has taught students basic cell and molecular biology techniques in an exciting research‐based format while instructing them in proper experimental design and data analysis.