An investigation of the effect of membrane curvature on transmembrane-domain dependent protein sorting in lipid bilayers

Abstract

Sorting of membrane proteins within the secretory pathway of eukaryotic cells is a complex process involving discrete sorting signals as well as physico-chemical properties of the transmembrane domain (TMD). Previous work demonstrated that tail-anchored (TA) protein sorting at the interface between the Endoplasmic Reticulum (ER) and the Golgi complex is exquisitely dependent on the length and hydrophobicity of the transmembrane domain, and suggested that an imbalance between TMD length and bilayer thickness (hydrophobic mismatch) could drive long TMD-containing proteins into curved membrane domains, including ER exit sites, with consequent export of the mismatched protein out of the ER. Here, we tested a possible role of curvature in TMD-dependent sorting in a model system consisting of Giant Unilamellar Vesicles (GUVs) from which narrow membrane tubes were pulled by micromanipulation. Fluorescent TA proteins differing in TMD length were incorporated into GUVs of uniform lipid composition or made of total ER lipids, and TMD-dependent sorting and diffusion, as well as the bending rigidity of bilayers made of microsomal lipids, were investigated. Long and short TMD-containing constructs were inserted with similar orientation, diffused equally rapidly in GUVs and in tubes pulled from GUVs, and no difference in their final distribution between planar and curved regions was detected. These results indicate that curvature alone is not sufficient to drive TMD-dependent sorting at the ER-Golgi interface, and set the basis for the investigation of the additional factors that must be required.

Keywords: :

• endoplasmic reticulum
• giant unilamellar vesicles
• hydrophobic mismatch
• nanotubes
• tail-anchored proteins
• optical tweezers
• bending rigidity

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Work performed in the laboratory of NB was supported by a grant from the Italian Cancer Association (AIRC, Investigator Grant 2009). MF was a doctoral student of the University of Milan and recipient of a short-term EMBO fellowship for collaborative work at the Institut Curie. We thank Sara Colombo for helpful discussions and support and the Fondazione Monzino (Milan) for its generous gift of the Zeiss LSM 510 Meta confocal microscope. We thank Patricia Bassereau for technical support and discussion, David Guet and Mathieu Pinot for assistance with the optical tweezers set-up and funding from Institut Curie, CNRS (Program ‘Prise de risque à l’interface Physique-Biologie’), ANR (grant number ANR09-JCJC-0020–01).

Supplementary Material

Supplementary material may be downloaded here: www.landesbioscience.com/journals/cellularlogistics/article/29087/