Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.

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The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

Mitochondria play a critical role in diverse cellular processes, such as energy production and apoptosis regulation. The mitochondria-targeted drug delivery is becoming a potential novel strategy for overcoming drug resistance in cancer therapy. Herein, we synthesize nature-inspired dopamine-derived polydopamine (PDA) nanoparticles. Using triphenylphosphonium (TPP) as the mitochondrial penetration molecule to improve the target efficiency, we synthesize poly(ethylene glycol) (PEG)-modified PDA (PDA-PEG) and TPP-functionalized PEG-modified PDA (PDA-PEG-TPP) nanoparticles. Then anticancer drug doxorubicin (DOX) was loaded on PDA-PEG and PDA-PEG-TPP (PDA-PEG-DOX and PDA-PEG-TPP-DOX) nanoparticles, which are apt to deliver DOX to cell nuclei and mitochondria, respectively. To mimic the repeated anticancer drug treatment in clinical cases, we repeatedly treated the MDA-MD-231 cancer cells for a long time using DOX-loaded nanoparticles and find that the mitochondria targeting PDA-PEG-TPP-DOX has higher potential to overcome the drug resistance than the regular delivery nanoparticles PDA-PEG-DOX. These results indicate the promising potential of applying PDA-PEG-TPP-DOX nanoparticles in mitochondria-targeted drug delivery to overcome the drug resistance in long-time anticancer chemotherapy.