期刊
JOURNAL OF CELL SCIENCE
卷 132, 期 6, 页码 -出版社
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.222315
关键词
CryoAPEX; Electron tomography; Membrane protein; FIC AMPylation; Cryofixation
类别
资金
- National Institute of General Medical Sciences of the National Institutes of Health [R01GM10092]
- Indiana Clinical and Translational Sciences Institute Research Grant [CTSI-106564]
- Purdue University Institute for Inflammation, Immunology, and Infectious Disease Core Start Grant [PI4D-209263]
We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.
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