Joyce E. Young

Phospholipid fatty acyl spatial distribution in bovine rod outer segment disk membranes

The distribution of fatty acids within the phospholipid headgroup classes was investigated as a function of the age/spatial distribution of bovine rod outer segment disk membranes. The disks were separated into subpopulations based upon the cholesterol content in their membranes. Because disk membrane cholesterol content decreases as the disks are apically displaced in the rod outer segment, this separation yields disk subpopulations of different ages and from age-dependent spatial locations within the outer segment. The phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), of each of these subpopulations were separated and the fatty acid composition of each was determined. These data indicated that while most of the fatty acids show little or no change with age/spatial location, some pronounced changes can be observed in certain classes. Within the PC class, 16:0 dramatically decreases with disk age while the 22:6 increases with disk age. While the PE class exhibits some fatty acid changes, they are small. The PS class exhibits no significant changes in fatty acid composition. The PI class which constitutes less than 2% of the total phospholipid exhibits age-related changes in each of the fatty acids which could be measured. Most notable of these is an increase in 20:4 as the disks are apically displaced. These changes indicate a remodeling of the disk membranes which may be related to the phototransduction process or to preparation for eventual disk phagocytosis.

Inhibition of sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures.(ABSTRACT TRUNCATED AT 400 WORDS)

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Retinal and retinol promote membrane fusion

Disk membranes from the bovine retinal rod outer segments (ROS) were found to fuse with vesicles made of lipids extracted from unbleached ROS disk membranes, using a lipid mixing assay for membrane fusion (relief of self-quenching of R18, octadecylrhodamine B chloride). If the retinal chromophore of rhodopsin was reductively linked to opsin before lipid extraction, the vesicles made of the extracted lipids were not suitable targets for fusion of the disk membranes. The addition of retinal and retinol to these vesicles restored their ability to fuse. Therefore, the presence of all-trans retinal was implicated in promoting membrane fusion in this system. To test this possibility, the ability of retinal and retinol to influence the phase behavior and the fusion capability of large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl-DOPE) was examined. Both retinal and retinol stimulated the fusion of vesicles of N-methyl-DOPE (contents mixing with ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX, p-xylylene bis(pyridinium bromide)). Both compounds reduced the onset temperature for isotropic resonances in the 31P-NMR spectra of N-methyl-DOPE dispersions and the onset temperature, TH, for formation of hexagonal II phase. These results were consistent with previous studies in which the onset temperature for the 31P-NMR isotropic resonances were correlated with stimulation of membrane fusion. These data suggested that both retinal and retinol may stimulate membrane fusion by destabilizing the bilayers of membranes.

Effect of G Protein-Coupled Receptor Kinase 1 (Grk1) Overexpression on Rod Photoreceptor Cell Viability

PURPOSE Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

Inhibition of Sendai Virus Fusion and Phospholipid Vesicle Fusion: Implications for the Pathway of Membrane Fusion

Membrane fusion is an essential step in the infection cycle of enveloped viruses. Fusion may occur with the plasma membrane or may occur by endocytosis of the virion followed by acidification and subsequent pH-induced fusion. Enveloped viruses possess an outer limiting membrane containing glycoproteins responsible for recognition of the target cell and mediation of the fusion event. Fusion may be facilitated by a dedicated protein, such as the F protein of Sendai, or the ability to facilitate fusion and the binding of the virion to the target membrane may involve a single protein species. Fusion of the viral envelope with the target membrane allows the entry of the viral genome and initiation of replication. At present an adequate understanding of the mechanism of the viral membrane fusion process is lacking.