期刊
JOURNAL OF BIOMEDICAL OPTICS
卷 17, 期 1, 页码 -出版社
SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.JBO.17.1.011008
关键词
Forster resonance energy transfer; fluorescence recovery after photobleaching; diffusion; protein-protein interactions; systems biology; live cells; green fluorescent protein; fluorescence microscopy
资金
- [R01 GM073846]
- [3R01 GM73846-4S1]
- [NSF/DMS 0970008]
- Division Of Mathematical Sciences
- Direct For Mathematical & Physical Scien [0970008] Funding Source: National Science Foundation
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 Forster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4B(C74A), 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 Atg4B(C74A) 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 Atg4B(C74A) 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 Atg4B(C74A) and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells. (c) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.17.1.011008]
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据