Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis.

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UVvisible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind alltrans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C 15 -syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.Living cells react to stimuli, which are realized in physical or chemical signals and are often detected by specialized membrane receptor proteins. G-protein-coupled receptors (GPCRs) 1 transmit their signal to heterotrimeric G-proteins via cytoplasmic domains of their seven-transmembrane ␣-helical structure. The majority of signals are chemical ligands, such as hormones or pheromones, which reach their GPCR by diffusion and bind to a site near their extracellular surface (1, 2). Photoreceptors contain a chromophore as a fixed prostethic group and are specialized for the detection of quanta of visible light in the environment (3, 4). The first step in the signal transduction pathway mediated by these receptors is the generation of a short-lived electronically excited state caused by photon absorption to channel the energy into a conformationally and/or chemically altered state of chromophore-protein interaction (5). Although these early events of light absorption are lacking in ligand binding receptors, G-protein-coupled photoreceptors may use similar mechanisms to spread the local activation near the ligand binding site to the cytoplasmic binding sites, where the G-protein has access. In this view, the chromophore can be understood as a fixed ligand that becomes an agonist by light absorpt...

Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G t ). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G t with Meta III, which was previously considered inactive. G t thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.The visual pigment in retinal rods, rhodopsin, is an archetype of a G-protein-coupled receptor and belongs to the large class I of the G-protein-coupled receptor superfamily. Its chromophoric ligand, 11-cis-retinal, is responsible for the absorption of light and for the function as a photoreceptor. To make the receptor permanently available for light excitation, the retinal is bound to the opsin apoprotein by a covalent Schiff base linkage. However, in contrast to other retinal proteins, which serve biological functions from proton transport to archaeal phototaxis, the Schiff base is not unconditionally stable during the whole lifetime of the protein (see Ref. 1). Within minutes after light excitation and during the lifetime of the active, G-protein binding conformation, the linkage to the photoisomerized chromophore is broken by hydrolysis, and the photolyzed all-trans-retinal is released from the active site (see Refs. 2 and 3). The site is then filled with fresh 11-cis-retinal delivered by a specialized retinal metabolism that involves a complex enzymology in the distant cells of the pigment epithelium (see Ref. 4). This so-called retinoid cycle is necessary because the retinal exchange mechanism in rhodopsin is exclusive and unidirectional, so that not even the absorption of blue light allows the restoration of the original 11-cis-retinal opsin complex. This is likely to be the price to be paid for the exceedingly stable ground state provided by the tight packing of this form of retina...