Abstract
We describe a method, termed cryoAPEX, that 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/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/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.
Summary statement CryoAPEX couples localization of peroxidase-tagged membrane proteins at high-resolution with 3D structural analysis, within an optimally preserved cellular context.
