Quantitative analysis of membrane protein transport across the nuclear pore complex.
Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG-Nups in the central channel, and the linker can position the transport factor-bound NLS in the vicinity of the FG-Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin-mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC.
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Anne C
AC
Meinema
Bert
B
Poolman
Liesbeth M
LM
Veenhoff
D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D004058:Diffusion; D017930:Genes, Reporter; D028884:Karyopherins / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals; D022022:Nuclear Pore / Q000378:metabolism*; D009687:Nuclear Proteins / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
C556514:Heh2 protein, S cerevisiae; D028884:Karyopherins; D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins