Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin ␣/␤.Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257 GKISKH-WTGI 266 . This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin ␣. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin ␣, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin ␣.Nuclear transport is an active signal-mediated process that requires, in most cases, soluble transport factors and specific import signals. Two families of transport receptors have been identified in the importin ␤ superfamily, which is involved in both nuclear import and export, and the TAP superfamily that mediates nuclear export. Transport receptors recognize specific nuclear localization signals (NLSs) 1 and nuclear export signals exposed on the molecular surface of cargoes. In the classical nuclear import pathway proteins bearing a classical SV40-like NLS (PKKKRKV) are recognized by the importin ␣/␤ heterodimer (also known as karyopherin ␣/␤) (1-3). Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6, 7

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated retinal release from a short-wavelength sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t1/2) of retinal release from VCOP was 7.1 s, 250-fold faster than rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t1/2 decreasing from 23 s to 4 s with pH 4.1 to 8, respectively. However, the Arrhenius activation energy (Ea) for VCOP derived from kinetic measurements between 4° and 20°C was 17.4 kcal/mol, similar to 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D2O) effect in VCOP, but less than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOPD108A) produced a pigment with an unprotonated chromophore (⌊max = 360 nm) and dramatically slowed (t1/2 ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D/ D108A) was designed to move the counterion one alpha helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (⌊max = 420 nm). Moreover, VCOPS85D/D108A mutant had retinal release kinetics (t1/2 = 7 s) and Ea (18 kcal/mol) similar to the native pigment exhibiting no pH-dependence. By contrast, the single mutant VCOPS85D had a ~3-fold decrease in retinal release rate compared to the native pigment. Photoactivated VCOPD108A had kinetics comparable to a rhodopsin counterion mutant, RhoE113Q, both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis, but rather from differences in the non-covalent binding properties of the retinal chromophore to the protein.

Assignment of the protonation state of the residue Glu-181 is important to our understanding of the primary event, activation processes and wavelength selection in rhodopsin. Despite extensive study, there is no general agreement on the protonation state of this residue in the literature. Electronic assignment is complicated by the location of Glu-181 near the nodal point in the electrostatic charge shift that accompanies excitation of the chromophore into the low-lying, strongly allowed ππ* state. Thus, the charge on this residue is effectively hidden from electronic spectroscopy. This situation is resolved in bathorhodopsin, because photoisomerization of the chromophore places Glu-181 well within the region of negative charge shift following excitation. We demonstrate that Glu-181 is negatively charged in bathorhodopsin based on the shift in the batho absorption maxima at 10K [λ max band (native)= 544±2 nm, λ max band (E181Q)= 556±3 nm] and the decrease in the λ max band oscillator strength (0.069±0.004) of E181Q relative to the native protein. Because the primary event in rhodopsin does not include a proton translocation or disruption of the hydrogen-bonding network within the binding pocket, we may conclude that the Glu-181 residue in rhodopsin is also charged.Rhodopsin is a membrane bound photoreceptor protein responsible for scotopic (dim light) vision in humans and animals with image resolving eyes. Rhodopsin is the first G protein coupled receptor (GPCR) for which a crystal structure was obtained. 1, 2 The protein consists of seven transmembrane α-helices and an 11-cis retinal chromophore covalently bound via a protonated Schiff based linkage to Lys-296. The primary photochemical event generates the intermediate bathorhodopsin (batho), which is stable at low temperatures and contains an all-trans chromophore. 3 Thermal decay of batho generates a series of less energetic intermediates (BSI, Lumi, Meta I and Meta II). The Meta II intermediate is responsible for activating the heterotrimeric G-protein, transducin, which in turn initiates the visual signal cascade. 4, 5 Because GPCRs comprise the largest protein family in the human genome, a greater understanding of the activation pathway is important to drug discovery and development.6 Elucidation of the photoactivation mechanism of rhodopsin should yield insight into the activation pathway of all class A GPCRs. Recently, a new mechanism of rhodopsin activation has been proposed based on the observation of a counterion-switch during the photobleaching sequence. 7 Subsequent studies of cone pigments indicate that a counterion switch also occurs in the blue and ultraviolet cone pigments. 8,9 These studies support the concept that a counterion switch may be a generic requisite for GPCR activation. 7,10 The basic elements of the counterion switch can be understood by reference to Figure 1. Glu-113, the primary counterion in the dark state, forms a water mediated salt bridge with the imine linkage of the protonated Schiff base of the 11-cis retinal chrom...

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.