CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins.
J Cell Sci
; 132(6)2019 03 18.
Article
en En
| MEDLINE
| ID: mdl-30886003
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tomografía con Microscopio Electrónico
/
Proteínas de la Membrana
Límite:
Humans
Idioma:
En
Revista:
J Cell Sci
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos