Abstract
Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples.We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample’s copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.
| Original language | English |
|---|---|
| Article number | e20210106 |
| Journal | Journal of Cell Biology |
| Volume | 220 |
| Issue number | 8 |
| DOIs | |
| State | Published - 2 Aug 2021 |
| Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2021 Sander et al.
Funding
We thank David Peck, Tim Dinh, and Dr. Huajun Yan for isolation of the bovine retinas. We also thank Dr. Brian Kevany for help with the initial nanobody expression and screening. The XIRAN-series SMA was provided by Polyscope Polymers B.V. The CL2 antibody was continuously produced by Denice Major at the Visual Science Research Core of Case Western Reserve University (Cleveland, OH), supported by P30 EY11373. This research was supported in part by grants to K. Palc-zewski from the National Institutes of Health (NIH; EY009339, EY027283, EY030873, and EY019312); and to P.D. Kiser from the U.S. Department of Veterans Affairs (I01BX004939). D. Skowronska-Krawczyk was supported by a grant from the Thome Memorial Foundation Program in Age-Related Macular Degeneration Research. C.L. Sander was supported by National Eye Institute–funded predoctoral fellowships T32EY007157-17 and T32EY007157-16A1. E.H. Choi was supported by predoctoral fellowships T32GM007250 and T32GM008803. S. Suh was supported by predoctoral fellowships F30EY029136-01A1, T32EY024236, and T32GM007250. The authors also acknowledge support from a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology, University of California, Irvine. This work was also supported by the Mass Spectrometry Core of the Salk Institute with funding from NIH-National Cancer Institute Cancer Center Support Grant (P30 014195) and the Helmsley Center for Genomic Medicine. The MS data described here were gathered on a Thermo Fisher Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer funded by National Institutes of Health grant 1S10OD021815-01. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health grant P41-GM103311.
| Funders | Funder number |
|---|---|
| Helmsley Center for Genomic Medicine | 1S10OD021815-01, P41-GM103311 |
| Mass Spectrometry Core of the Salk Institute | |
| Visual Science Research Core of Case Western Reserve University | P30 EY11373 |
| National Institutes of Health | EY019312, EY030873, EY009339 |
| National Eye Institute | T32GM008803, T32GM007250, T32EY024236, R24EY027283, T32EY007157-16A1, F30EY029136-01A1 |
| National Cancer Institute | P30 014195 |
| U.S. Department of Veterans Affairs | I01BX004939 |
| Research to Prevent Blindness | |
| Edward N. and Della L. Thome Memorial Foundation | |
| University of California, Irvine |