CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins

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.

Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo

“…On a molecular level, mFICD deficiency depletes unstressed cells of most AMPylated proteins. In accordance with previous studies, we find evidence that mFICD AMPylates both ER-resident and cytoplasmic proteins ( 11 , 19 , 20 , 25 , 26 , 27 , 28 , 29 , 31 , 39 ). The mechanisms that underlie mFICD's ability to target topologically distinct compartments remain to be defined.…”

“…In addition to BiP, fic AMPylases also modify a wide range of non-ER proteins ( 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ). Indeed, fic AMPylases are also present in the nuclear envelope and the cytoplasm ( 11 , 20 , 28 ).…”

Alpha-Synuclein is a Target of Fic-mediated Adenylylation/AMPylation: Implications for Parkinson’s Disease

“…However, some cellular and proteomic analyses have identified additional targetssuch as histones and cytosolic Hsp70 and Hsp40 family chaperones -that localize to cellular compartments other than the ER [30], [31], [40]. How HYPE interacts physiologically with these non-ER localized proteins is unclear [54], though a recent report reveals that a mass exodus of ER resident proteins occurs in response to ER Ca 2+ depletion [41]. Additionally in C. elegans, HYPE is reported to be partially present in the cytosol in addition to the ER lumen [29].…”

AMPylation is a specific lysosomal protein posttranslational modification in neuronal maturation

“…FICD is characterized by its endoplasmic reticulum (ER) localization and dual catalytical activity of AMPylation and deAMPylation. (Preissler et al, 2017; Sengupta et al, 2019) FICD catalyzes an AMP transfer from its substrate ATP and reverses the modification by the hydrolysis of the AMP-protein ester. FICD’s catalytic activity is regulated by its α-helix inhibition loop through the interaction of Glu234 positioned in the inhibition loop and Arg374, which is necessary for a complexation of ATP in the active site (Engel et al, 2012).…”