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3 February 2012 Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching
Lewis Kraft, Anne Kenworthy
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Funded by: FRET standards, EGFP-LC3, Cerulean and Venus constructs
Journal of Biomedical Optics, Vol. 17, Issue 1, 011008 (February 2012). https://doi.org/10.1117/1.JBO.17.1.011008
Abstract
The protein microtubule-associated protein 1, light chain 3 (LC3) functions in autophagosome formation and plays a central role in the autophagy pathway. Previously, we found LC3 diffuses more slowly in cells than is expected for a freely diffusing monomer, suggesting it may constitutively associate with a macromolecular complex containing other protein components of the pathway. In the current study, we used Förster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4BC74A, a catalytically inactive mutant of the cysteine protease involved in lipidation and de-lipidation of LC3, as a model system to probe protein complex formation in the autophagy pathway. We show Atg4BC74A is in FRET proximity with LC3 in both the cytoplasm and nucleus of living cells, consistent with previous biochemical evidence that suggests these proteins directly interact. In addition, overexpressed Atg4BC74A diffuses significantly more slowly than predicted based on its molecular weight, and its translational diffusion coefficient is significantly slowed upon coexpression with LC3 to match that of LC3 itself. Taken together, these results suggest Atg4BC74A and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells.
© 2012 Society of Photo-Optical Instrumentation Engineers (SPIE) 0091-3286/2012/$25.00 © 2012 SPIE
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Lewis Kraft and Anne Kenworthy "Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching," Journal of Biomedical Optics 17(1), 011008 (3 February 2012). https://doi.org/10.1117/1.JBO.17.1.011008
Published: 3 February 2012
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KEYWORDS
Fluorescence resonance energy transfer
Proteins
Venus
Diffusion
Luminescence
Confocal microscopy
Molecules
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