Glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins, such as Gce1, the dually acylated nonreceptor tyrosine kinases (NRTKs), such as pp59 Lyn , and the membrane protein, caveolin, together with cholesterol are typical components of detergent/carbonate-insoluble glycolipidenriched raft domains (DIGs) in the plasma membrane of most eucaryotes. Previous studies demonstrated the dissociation from caveolin and concomitant redistribution from DIGs of Gce1 and pp59 Lyn in rat adipocytes in response to four different insulin-mimetic stimuli, glimepiride, phosphoinositolglycans, caveolin-binding domain peptide, and trypsin/NaCl-treatment. We now characterized the structural basis for this dynamic of DIG components. Materials and Methods: Carbonate extracts from purified plasma membranes of basal and stimulated adipocytes were analyzed by high-resolution sucrose gradient centrifugation. Results: This process revealed the existence of two distinct species of detergent/carbonate-insoluble complexes floating at higher buoyant density and harboring lower amounts of cholesterol, caveolin, GPI proteins, and NRTKs (lcDIGs) compared to typical DIGs of high cholesterol content (hcDIGs). The four insulin-mimetic stimuli decreased by 40-70% and increased by 2.5-to 5-fold the amounts of GPI proteins and NRTKs at hcDIGs and lcDIGs, respectively.
The ABC-transporter HlyB is a central element of the Type I protein secretion machinery, dedicated to export the E. coli toxin HlyA in a single step across the two membranes of the cell envelope. Here, we discuss recent insights into the structure and the mechanism of ATP-hydrolysis by the NBD of HlyB. Combining structural and biochemical data, we have suggested that substrate-assisted catalysis (SAC), but not general base catalysis, is responsible for ATP-hydrolysis in this NBD and might also operate in other NBDs. Finally, the implications and advantages of SAC are discussed in the context of ATP-induced dimerization of the NBDs.
Background: Fungal multidrug efflux pumps possess a degenerate nucleotide-binding site (NBS). Results: Restoring all the nonconserved amino acids in the degenerate NBS of Pdr5 leads to complete loss of function of the protein.
Conclusion:The degenerate NBS is essential and acts as a structural platform supporting the canonical NBS. Significance: This is the first study dealing with the entire degenerate NBS and its functional role.
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