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 co-immunopurify 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 were 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 provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS.
SUMMARY Sander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- ABC
- ATP-binding cassette
- AcCa
- acylcarnitine
- AMP
- adenosine monophosphate
- ATR
- all-trans retinal
- BTP
- bis-tris propane
- CDR
- complimentary determining regions
- Cer
- ceramides
- ChE
- cholesterol ester
- CHS
- cholesterol hemisuccinate
- CMC
- critical micelle concentration
- CNBr
- cyanogen bromide
- cryoTEM
- cryogenic transmission electron microscopy
- DDM
- n-dodecyl â-D-maltoside
- DG
- diacylglycerol
- DHA
- docosahexaenoic acid
- DIBMA
- diisobutylene maleic acid
- DIBMALP
- diisobutylene maleic acid lipid particles
- ECD
- extracytosolic domain
- EW
- elution wash
- FA
- fatty acid
- FFA
- free fatty acid
- FT
- flow-through
- FWR
- framework regions
- GPCR
- G protein-coupled receptor
- H1/2/3
- hypervariable regions or loops 1/2/3
- IHC
- immunohistochemistry
- kDa
- kilodalton
- KLH
- keyhole limpet hemocyanin
- KO
- knock-out
- L
- load
- LC-MS
- liquid chromatography-mass spectrometry
- LC-PUFA
- long chain-polyunsaturated fatty acid
- LMNG
- laurel maltose neopentyl glycol
- LPA
- lyso-phosphatidic acid
- LPC
- lyso-phosphatidylcholine
- LPE
- lyso-phosphatidylethanolamine
- LUV
- large unilamillar vesicles
- lyso-PL
- lyso-phospholipid
- mAb
- monoclonal antibody
- MG
- monoacylglycerol
- MS
- mass spectroscopy
- MSP
- membrane scaffold protein
- Nb
- nanobody
- N-ret-PE
- N-retinylidene-phosphatidylethanolamine
- nsTEM
- negative stain transmission electron microscopy
- PA
- phosphatidic acid
- PAGE
- polyacrylamide gel electrophoresis
- PBS
- phosphate-buffered saline
- PBST
- phosphate-buffered saline with Tween-20
- PC
- phosphatidylcholine
- PE
- phosphatidylethanolamine
- PRPH2
- peripherin2
- PI
- phosphatidylinositol
- PS
- phosphatidylserine
- PVDF
- polyvinylidene difluoride
- Res
- resin
- ROM1
- rod outer segment membrane protein 1
- ROS
- rod outer segment
- RPE65
- retinal pigment epithelium-specific 65 kDa protein
- RT
- room temperature
- SEC
- size exclusion chromatography
- SMA
- styrene maleic acid
- SMALP
- styrene maleic acid lipid particle
- SDS
- sodium dodecyl sulfate
- TCEP
- tris(2-carboxyethyl)phosphine)
- TG
- triacylglycerol
- TMD
- transmembrane domain
- VLC-PUFA
- very long chain-polyunsaturated fatty acid
- W1-4
- wash 1-4
- WT
- wild-type