The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCPO to the red OCPR form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP’s N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.

Proteorhodopsin (PR), a light-driven proton pump from marine proteobacteria, exhibits photocycle characteristics similar to bacteriorhodopsin (BR) at neutral pH, including an M-like photointermediate. However, at acidic pH, spectroscopic evidence for an M-like species was absent, and the vectoriality of proton pumping was inverted. To gain further insight into this unusual property, we examined the voltage dependence of stationary and laser flash-induced photocurrents of PR under different pH conditions upon expression in Xenopus oocytes. The current–voltage curves were linear under all conditions tested, and photocurrent reversal potentials distinctly depended on the pH gradient. PR mutants D97N and D97T exhibited transient and stationary inward currents already at neutral pH, showing that neutralization of the proton acceptor abolishes forward pumping and permits only inward proton transport. Mutation E108G, which disrupts the donor site for Schiff base (SB) reprotonation, resulted in largely reduced photocurrents, which could be strongly stimulated by azide, similar to previous observations on BR mutant D96G. When PR and BR photocurrents in response to blue or green laser flashes during or after continuous illumination were compared, direct electrical evidence for the occurrence of an M-like intermediate at neutral pH could only be obtained when reprotonation of the SB was slowed down by PR mutation E108G. For PR at acidic pH, laser flashes only produced inwardly directed photocurrents, independent from background illumination, thus precluding electrical identification of an M-like species. However, when visible absorption spectroscopy was carried out at low temperatures, occurrence of an M-like species was robustly observed at low pH. This indicates that SB deprotonation and reprotonation occur during the PR photocycle also at low pH. Our results corroborate the conclusion that in PR, the direction of proton pumping can be switched by changes in pH and membrane potential, with the protonation state of Asp-97 being the key determinant for selecting between transport modes.