Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication

• Published in: The EMBO journal Vol. 30; no. 16; pp. 3337 - 3352
• Main Authors: Kupke, Thomas, Di Cecco, Leontina, Müller, Hans-Michael, Neuner, Annett, Adolf, Frank, Wieland, Felix, Nickel, Walter, Schiebel, Elmar
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 17.08.2011; Nature Publishing Group UK; Blackwell Publishing Ltd; Nature Publishing Group
• Subjects: Active Transport, Cell Nucleus - physiology; Amino Acid Motifs; Amino Acid Sequence; amphipathic helix; beta Karyopherins - genetics; beta Karyopherins - physiology; Cell cycle; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cell Cycle Proteins - physiology; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - genetics; Cytoskeletal Proteins - physiology; EMBO05; EMBO06; in-plane membrane anchor; Kap123; Liposomes - metabolism; Membrane Fusion; Membranes; Molecular biology; Molecular Sequence Data; Nbp1; Nuclear Envelope - metabolism; Nuclear Localization Signals; Nuclear Pore Complex Proteins - metabolism; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - physiology; Phosphatidylcholines - metabolism; Protein Binding; Protein Interaction Mapping; Protein Structure, Secondary; Proteins; Recombinant Fusion Proteins - physiology; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Sequence Deletion; Signal transduction; Spindle Apparatus - metabolism; spindle pole body duplication; Structure-Activity Relationship; spindle pole body duplication; Nbp1; amphipathic helix; in‐plane membrane anchor; Kap123
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2011.242

Spindle pole bodies (SPBs), like nuclear pore complexes, are embedded in the nuclear envelope (NE) at sites of fusion of the inner and outer nuclear membranes. A network of interacting proteins is required to insert a cytoplasmic SPB precursor into the NE. A central player of this network is Nbp1 that interacts with the conserved integral membrane protein Ndc1. Here, we establish that Nbp1 is a monotopic membrane protein that is essential for SPB insertion at the inner face of the NE. In vitro and in vivo studies identified an N‐terminal amphipathic α‐helix of Nbp1 as a membrane‐binding element, with crucial functions in SPB duplication. The karyopherin Kap123 binds to a nuclear localization sequence next to this amphipathic α‐helix and prevents unspecific tethering of Nbp1 to membranes. After transport into the nucleus, Nbp1 binds to the inner nuclear membrane. These data define the targeting pathway of a SPB component and suggest that the amphipathic α‐helix of Nbp1 is important for SPB insertion into the NE from within the nucleus. How microtubule‐organizing spindle pole bodies are inserted into the nuclear envelope remains incompletely understood. This work reveals a role for a membrane‐active amphipathic helix in a key factor, whose masking by karyopherins ensures that insertion takes place from the inner face of the nuclear membrane.