title: Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.
abstract: Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France Ecole Doctorale Gènes Génomes Cellules, Université Paris Sud, F-91405 Orsay, France.
authors.forename: Aurélie G
authors.initials: AG
authors.lastname: Floch
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France Membrane Traffic in Health & Disease, INSERM ERL U950, 75013 Paris, France.
authors.forename: David
authors.initials: D
authors.lastname: Tareste
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France.
authors.forename: Patrick F J
authors.initials: PF
authors.lastname: Fuchs
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France Ecole Doctorale Gènes Génomes Cellules, Université Paris Sud, F-91405 Orsay, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Chadrin
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France.
authors.forename: Ikrame
authors.initials: I
authors.lastname: Naciri
authors.affiliation: Proteomic facility, Institut Jacques Monod, F-75205 Paris, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Léger
authors.affiliation: Universität des Saarlandes, Medizinische Biochemie und Molekularbiologie, D-66421 Homburg, Germany.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Schlenstedt
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Palancade
authors.affiliation: Institut Jacques Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France doye.valerie@ijm.univ-paris-diderot.fr.
authors.forename: Valérie
authors.initials: V
authors.lastname: Doye
mesh_terms: D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D015966:Gene Expression Regulation, Fungal; D008055:Lipids / Q000235:genetics; D008081:Liposomes / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D022022:Nuclear Pore / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: C109173:Kap123 protein, S cerevisiae; D008055:Lipids; D008081:Liposomes; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; C550848:Pom33 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins
keywords: Amphipathic helix; Integral membrane protein; Karyopherin; NPC; NPC distribution; NPC targeting; Nuclear pore complex; TMEM33 family