The acquisition of the tertiary and quartenary structures of newly synthesized soluble proteins is facilitated by molecular chaperones. Nothing is known, however, about how an integral membrane protein (IMP) acquires its tertiary structure. Are chaperone-like molecules involved? If so, where are they localized? Using a blot-type assay and a co-immunoprecipitation method, the investigator has determined that an IMP (p75) of Neurospora endoplasmic reticulum (ER) binds to mutant proteins of the H+ATPase, an IMP. Pulse-chase experiments will be conducted to determine the kinetics of the transient association of p75 with newly synthesized H+ATPase. p75 will be localized in situ using immunofluorescence and immunogold labeling of cryosections of Neurospora. Also, the genomic DNA that encodes p75 will be cloned and sequenced. Using this clone, the allele for p75 will be disrupted, using a technique called RIP, and the mutant phenotype will be investigated. If successful, these studies could provide evidence for a chaperone-like molecule that facilitates the proper folding of a cation-motive ATPase. These studies should also provide insight into the quality-control mechanisms of the ER and provide new opportunities to examine, and possibly modify, the export of proteins from the ER. Furthermore, the elucidation of the folding pathway of the H+ATPase will be extremely useful to other laboratories studying other cation-motive ATPases.
