Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks

Abstract

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

Fig. 1

(a) Representative Coomassie blue-stained 12% SDS–PAGE gel of bovine ROS membrane fractions [F1–F8, and pellet (P)] obtained by Triton X-100 treatment and differential sucrose density-gradient ultracentrifugation. (b) Semi-quantitative western (immunoblot) analysis of the distribution of Cav-1 (upper panel) and a representative Cav-1 immunoblot (lower panel) of these membrane fractions. Migration positions of protein molecular weight markers are denoted by numbers on the left-hand side of the gel and blot. Cav-1 distribution was determined by densitometric analyses of blots; the amount in each fraction is plotted as a percent of the total Cav-1 in all the fractions (see Materials and methods). Error bars represent standard deviation (SD) of the mean percent in each fraction (N = 4 DRM fractionations, from 4 independent ROS preparations). (c) Coomassie blue-stained 12% SDS–PAGE gel of proteins from concentrated DRM fractions subsequently analyzed by mass spectrometry. Band numbers of excised bands subjected to mass spectrometric analysis are indicated to the right of the gel. These band numbers correspond to those indicated in Table 1.

Fig. 2

Characterization of tryptic peptides from bovine DRM proteins using MALDI-TOF mass spectrometry. (a) MS spectrum of the tryptic digests using MALDI-TOF. The peaks with the assigned m/z value consistent with the theoretical tryptic peptide mass of the candidate CNG-α1 subunit are indicated by the asterisks. (b) MS/MS spectrum of an ion peak at m/z 1181.55 (see panel a) using MALDI-QIT-TOF MS. The C-terminal y-series and N-terminal b-series ions derived from the peptide corresponded to the amino acid residues 261–269 of CNGα-1.

Fig. 3

Semi-quantitative analysis of the fractionation of integral membrane proteins to ROS-derived DRMs. DRM fractions (F1–F4) contain only a small percentage of the total amount of ABCA4 (a), a somewhat higher percentage of rod opsin (b), and dramatic enrichment of Glut-1 (c) and CNG β-1 (d). In each case, the upper panels show the percentage of each protein (solid lines) plotted along with the distribution of Cav-1 (dashed lines, replotted from Fig. 1) to designate DRM fractions; lower panels show corresponding western blot. Error bars represent the SD of mean values obtained from four independent DRM preparations (from 4 separate ROS preparations).

Fig. 4

Dark-field, light microscopic, silver-enhanced immunogold labeling of bovine retina with antibodies against (a) Cav-1 and (b) Glut-1. In each, the inset panel represents a higher-magnification image. Note the minimal, diffuse labeling of the outer segment (OS) layer with both DRM-specific markers. Abbreviations: IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OLM, outer limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; BV, blood vessel. Scale bar (all panels): 25 μm.

Fig. 5

Isolation and characterization of DRM and non-DRM fractions from bovine ROS plasma membranes (PM) and disks. (a) Upper panel: GelCode Blue®-stained SDS–PAGE gel of isolated ROS PM and disk fractions. Lower panel: Corresponding western blots, probed with antibodies to disk marker proteins (CNGβ-1 and Rds), a PM marker protein in disks (Glut-1), and a DRM marker protein (Cav-1). (b) Western blot analysis of sucrose gradient fractions of DRMs prepared from ROS PM, probed with antibodies against CNGβ-1, rod opsin, Glut-1, and Cav-1. (c) Western blot analysis of sucrose gradient fractions of DRMs prepared from ROS disk membranes probed with antibodies against CNGβ-1, rod opsin, Rom-1, and Cav-1. Note that the DRM fraction distribution in PM is shifted to slightly heavier density (F4–F5), compared to the DRM fractions obtained from disk membranes (F3–F4).

Fig. 6

Semi-quantitative analyses of rod opsin distribution in DRM fractions derived from disk and PM-enriched membranes. (a) Distributions of rod opsin in DRM fractions from disks (filled squares/solid line) and PM (open circles/dotted line) were determined by densitometric analyses of blots from three independent DRM fractionations. The amount of rod opsin in each fraction is plotted as a percent of the total rod opsin in all the fractions. Error bars represent standard deviation (S.D.) of the mean percent in each fraction (N = 3 DRM fractionations each from three independent disk/PM isolations). Representative immunoblots from disk (b) and from PM (c) are also shown.

Fig. 7

(a) Cholesterol/phospholipid (Chol: PL) mole ratio of unfractionated ROS disk membranes, disk DRMs, unfractionated ROS plasma membranes (PM), and PM-derived DRMs. (b) Correlative distribution (relative mol%) of saturated (black bars), monounsaturated (shaded bars), and polyunsaturated (open bars) fatty acids in each of the membrane fractions shown in panel a. Data represent mean values (with SD error bars), N = 3 independent preparations each. Asterisks (*) denoted statistically significant differences (p < 0.05).

Fig. 8

Comparative fatty acid composition analysis of ROS disk and PM fractions (mean values expressed as relative mol%, with SD; N = 3). (a) Disk membranes (black bars) vs. plasma membranes (shaded bars). (b) Unfractionated disk membranes (black bars) vs. disk DRMs (shaded bars). (c) Unfractionated plasma membranes (black bars) vs. plasma membrane DRMs (shaded bars). (d) Disk DRMs (black bars) vs. plasma membrane DRMs (shaded bars). Asterisks (*) denoted statistically significant differences (p < 0.05).

Fig. 9

Schematic model depicting the segregation of ROS disk and PM proteins into DRM vs. non-DRM (bulk phase) domains. DRM domains are indicated by red circles. Rod opsin is entirely localized to the bulk phase in the ROS PM and is largely localized to the bulk phase in disk membranes, with a small portion residing in disk DRMs. Most Rom-1 and essentially all Rds and ABCA4 in the ROS are localized to non-DRM domains in disks; these proteins are not present in the ROS PM. The majority of CNG ion channels and essentially all Glut-1 transporters are localized to PM-derived DRMs; neither of these proteins is found in disks.