The Get1/2 insertase forms a channel to mediate the insertion of tail-anchored proteins into the ER
Paul Heo,
Jacob A. Culver,
Jennifer Miao,
Frederic Pincet,
Malaiyalam Mariappan
Affiliations
Paul Heo
Laboratoire de Physique de l’Ecole Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université de Paris, 75005 Paris, France; Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, 75014 Paris, France; Corresponding author
Jacob A. Culver
Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Nanobiology Institute, Yale University West Campus, West Haven, CT 06516, USA
Jennifer Miao
Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Nanobiology Institute, Yale University West Campus, West Haven, CT 06516, USA
Frederic Pincet
Laboratoire de Physique de l’Ecole Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université de Paris, 75005 Paris, France; Corresponding author
Malaiyalam Mariappan
Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Nanobiology Institute, Yale University West Campus, West Haven, CT 06516, USA; Corresponding author
Summary: Tail-anchored (TA) proteins contain a single C-terminal transmembrane domain (TMD) that is captured by the cytosolic Get3 in yeast (TRC40 in humans). Get3 delivers TA proteins to the Get1/2 complex for insertion into the endoplasmic reticulum (ER) membrane. How Get1/2 mediates insertion of TMDs of TA proteins into the membrane is poorly understood. Using bulk fluorescence and microfluidics assays, we show that Get1/2 forms an aqueous channel in reconstituted bilayers. We estimate the channel diameter to be ∼2.5 nm wide, corresponding to the circumference of two Get1/2 complexes. We find that the Get3 binding can seal the Get1/2 channel, which dynamically opens and closes. Our mutation analysis further shows that the Get1/2 channel activity is required to release TA proteins from Get3 for insertion into the membrane. Hence, we propose that the Get1/2 channel functions as an insertase for insertion of TMDs and as a translocase for translocation of C-terminal hydrophilic segments.
