A mutation in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) affecting endocytosis has been investigated at the cellular and protein structural levels. Quantitative examination of immunogold labeling of cytoskeletal whole mounts has shown that in cells expressing mutant GAPDH there is 1. more than a two-fold increase in the amount of that enzyme bound to microtubules; 2. a four-fold increase in the frequency of 24-45 nm knob-like protrusions from non-microtubule cytoskeletal filaments; 3. a four-fold increase in the amount of GAPDH on these knobs. The composition of these knobs is under investigation. Unlike the normal enzyme, mutant GAPDH is not displaced from microtubules by addition of ATP; paradoxically, enzymatic activity of mutant GAPDH is fifty times more sensitive to ATP, and binding of ATP to mutant enzyme is increased. Procedures have been worked out for covalent labeling of mutant and normal GAPDH with 8-azido-ATP32, proteolysis, and separation of peptides for sequencing, to identify the ATP binding sites on mutant and normal enzyme. This laboratory has taken a cell biological approach to investigation of the mechanism of action of FR901228, an anti-tumor bicyclic depsipeptide. Disassembly of the Golgi apparatus was observed to commence in cells treated with the drug for 2.5 hours. Golgi fragmentation was accompanied by a marked decrease in the rate of appearance of newly synthesized proteins on the plasma membrane. Recent results indicate that FR901228 effects an endoplasmic reticulum stress response prior to Golgi fragmentation. The G-protein linked calcium receptor in rMTC-44 cells has been examined by confocal microscopy. Location of the receptor was found to respond to extracellular calcium concentration: at 1.5 mM calcium, much of the receptor is intracellular, in large (0.5 - 1.0 micron) vesicles decorated with Gi proteins; on reduction of external calcium to 0.5 mM, receptor moves from the vesicles to the plasma membrane; restoring external calcium results in the rapid reappearance of the receptor in the vesicles.
