LISST

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This research program has as its goal understanding the structural basis for glycolytic enzyme co-localization and targeting at Z-discs and M- lines of the sarcomere as a means of understanding how this essential aspect of glycolytic enzyme organization provides the energy for muscle contraction. In the flight muscle of Drosophila, the glycolytic enzymes, glycerol-3-phosphate dehydrogenase (GPDH), glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and aldolase are localized at Z-discs/M- lines. Localization of GAPDH and aldolase depends on the simultaneous localization of GPDH. In the absence of glycolytic enzyme co- localization in flight muscles, flies can't fly. Further elucidation of the nature of protein-protein interactions that support colocalization will lead to a better understanding of the mechanisms by which ATP production from glycolysis supports muscle function. It remains to be determined whether glycolytic enzymes physically interact or whether other proteins play a role in co-localization. This will be determined using the yeast two hybrid system and co-immunoprecipitation techniques. The C-terminal tripeptide, Q-N, L found in the muscle specific isoform, GPDH-1 is required for GPDH targeting. Other domains of the glycolytic enzymes may play a role in targeting these enzymes to the Z-discs/M- lines and will be identified. Genes which encode proteins that interact with GPDH and aldolase and are functionally important in muscle function will be identified by selection of second site suppressor mutations. The developmental assembly process that results in co-localized glycolytic enzymes will be characterized. Targeting of additional glycolytic enzymes will be studied and one of these, aldolase, will be extensively developed. This will require isolation and phenotypic characterization of mutants in the aldolase gene. The role of the C- terminus in the aldolase muscle specific isoform will be evaluated by analyzing flight in transgenic flies which carry engineered transgenes. Finally, the details of the assembly process will be characterized by further development of an in vitro assay which uses isolated myofibrils as a platform on which to analyze the mechanism through which the glycolytic complex is assembled and organized at the Z-disc/M-line. The results of our work will have broad implications for understanding the connection between carbohydrate catabolism, energy production and muscle function. It is likely these implications will extend beyond the Drosophila model and should be relevant to understanding defects in human muscle function.