Absolute Quantification of Photoreceptor Outer Segment Proteins

Overview

Journal J Proteome Res

Publisher American Chemical Society

Specialty Biochemistry

Date 2023 Jul 26

PMID 37493966

Authors

Nikolai P Skiba

Tylor R Lewis

William J Spencer

Carson M Castillo

Andrej Shevchenko

Vadim Y Arshavsky

Affiliations

Soon will be listed here.

Abstract

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

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References

BOWNDS D, Robinson W . Characterization and analysis of frog photoreceptor membranes. J Gen Physiol. 1971; 58(3):225-37. PMC: 2226025. DOI: 10.1085/jgp.58.3.225. View

Shuford C, Walters J, Holland P, Sreenivasan U, Askari N, Ray K . Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised. Anal Chem. 2017; 89(14):7406-7415. DOI: 10.1021/acs.analchem.7b00858. View

Lyubarsky A, Daniele L, Pugh Jr E . From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG. Vision Res. 2004; 44(28):3235-51. DOI: 10.1016/j.visres.2004.09.019. View

Kaptan D, Penkov S, Zhang X, Gade V, Raghuraman B, Galli R . Exogenous ethanol induces a metabolic switch that prolongs the survival of Caenorhabditis elegans dauer larva and enhances its resistance to desiccation. Aging Cell. 2020; 19(10):e13214. PMC: 7576309. DOI: 10.1111/acel.13214. View

Bauer P, Drechsler M . Association of cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers in bovine retinal rod outer segment plasma membranes. J Physiol. 1992; 451:109-31. PMC: 1176153. DOI: 10.1113/jphysiol.1992.sp019156. View

Nickell S, Park P, Baumeister W, Palczewski K . Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography. J Cell Biol. 2007; 177(5):917-25. PMC: 2064290. DOI: 10.1083/jcb.200612010. View

Skiba N, Cady M, Molday L, Han J, Lewis T, Spencer W . TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane. Mol Cell Proteomics. 2021; 20:100088. PMC: 8167285. DOI: 10.1016/j.mcpro.2021.100088. View

El Mazouni D, Gros P . Cryo-EM structures of peripherin-2 and ROM1 suggest multiple roles in photoreceptor membrane morphogenesis. Sci Adv. 2022; 8(45):eadd3677. PMC: 9645710. DOI: 10.1126/sciadv.add3677. View

Lobanova E, Finkelstein S, Herrmann R, Chen Y, Kessler C, Michaud N . Transducin gamma-subunit sets expression levels of alpha- and beta-subunits and is crucial for rod viability. J Neurosci. 2008; 28(13):3510-20. PMC: 2795350. DOI: 10.1523/JNEUROSCI.0338-08.2008. View

10.

Klenchin V, Calvert P, Bownds M . Inhibition of rhodopsin kinase by recoverin. Further evidence for a negative feedback system in phototransduction. J Biol Chem. 1995; 270(27):16147-52. DOI: 10.1074/jbc.270.27.16147. View

11.

Peshenko I, Olshevskaya E, Savchenko A, Karan S, Palczewski K, Baehr W . Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors. Biochemistry. 2011; 50(25):5590-600. PMC: 3127287. DOI: 10.1021/bi200491b. View

12.

Kumar M, Has C, Lam-Kamath K, Ayciriex S, Dewett D, Bashir M . Vitamin A Deficiency Alters the Phototransduction Machinery and Distinct Non-Vision-Specific Pathways in the Eye Proteome. Biomolecules. 2022; 12(8). PMC: 9405971. DOI: 10.3390/biom12081083. View

13.

Hwang J, Lange C, Helten A, Hoppner-Heitmann D, Duda T, Sharma R . Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity. Eur J Biochem. 2003; 270(18):3814-21. DOI: 10.1046/j.1432-1033.2003.03770.x. View

14.

Lamb T . Photoreceptor physiology and evolution: cellular and molecular basis of rod and cone phototransduction. J Physiol. 2022; 600(21):4585-4601. PMC: 9790638. DOI: 10.1113/JP282058. View

15.

Caruso G, Klaus C, Hamm H, Gurevich V, Makino C, DiBenedetto E . Position of rhodopsin photoisomerization on the disk surface confers variability to the rising phase of the single photon response in vertebrate rod photoreceptors. PLoS One. 2020; 15(10):e0240527. PMC: 7556485. DOI: 10.1371/journal.pone.0240527. View

16.

Gulati S, Palczewski K . Structural view of G protein-coupled receptor signaling in the retinal rod outer segment. Trends Biochem Sci. 2022; 48(2):172-186. PMC: 9868064. DOI: 10.1016/j.tibs.2022.08.010. View

17.

Martemyanov K, Krispel C, Lishko P, Burns M, Arshavsky V . Functional comparison of RGS9 splice isoforms in a living cell. Proc Natl Acad Sci U S A. 2008; 105(52):20988-93. PMC: 2634932. DOI: 10.1073/pnas.0808941106. View

18.

Pentia D, Hosier S, Cote R . The glutamic acid-rich protein-2 (GARP2) is a high affinity rod photoreceptor phosphodiesterase (PDE6)-binding protein that modulates its catalytic properties. J Biol Chem. 2006; 281(9):5500-5. PMC: 2825572. DOI: 10.1074/jbc.M507488200. View

19.

Loewen C, Molday R . Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration. J Biol Chem. 2000; 275(8):5370-8. DOI: 10.1074/jbc.275.8.5370. View

20.

Zhong H, Molday L, Molday R, Yau K . The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry. Nature. 2002; 420(6912):193-8. PMC: 2877395. DOI: 10.1038/nature01201. View