title: Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane.
abstract: The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.
authors.affiliation: Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors.forename: Pascal
authors.initials: P
authors.lastname: Weber
authors.affiliation: Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Batoulis
authors.affiliation: Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany.
authors.forename: Kerstin M
authors.initials: KM
authors.lastname: Rink
authors.affiliation: Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Dahlhoff
authors.affiliation: Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Pinkwart
authors.affiliation: Heidelberg University Biochemistry Center (BZH), Heidelberg, Germany.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Söllner
authors.affiliation: Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors.forename: Thorsten
authors.identifier: 0000-0002-9128-0137
authors.initials: T
authors.lastname: Lang
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D054878:Lipoylation; D016716:PC12 Cells; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011994:Recombinant Proteins; C498858:Snap23 protein, rat; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; C042511:1,2-dioleoylphosphatidylserine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: SNAREs; biophysics; cell biology; liposome; membrane targeting; phosphoinositides; post-translational modification; protein electrostatics; rat; structural biology