title: Protein flexibility is required for vesicle tethering at the Golgi.
abstract: The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, United States.
authors.forename: Pak-yan Patricia
authors.initials: PY
authors.lastname: Cheung
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, United States.
authors.forename: Charles
authors.initials: C
authors.lastname: Limouse
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, United States.
authors.forename: Hideo
authors.initials: H
authors.lastname: Mabuchi
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, United States.
authors.forename: Suzanne R
authors.identifier: http://orcid.org/0000-0002-6462-984X
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011487:Protein Conformation
chemical_list: C472299:GCC2 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins
keywords: Golgi complex; biochemistry; cell biology; coiled-coil; human; membrane traffic; vesicle tethering