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In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex(More)
The molecular dynamics of nuclear protein import were examined in a solution binding assay by testing for interactions between a protein containing a nuclear localization signal (NLS), the transport factors karyopherin alpha, karyopherin beta, and Ran, and FXFG or GLFG repeat regions of nucleoporins. We found that karyopherins alpha and beta cooperate to(More)
The transport of pro-alpha-factor from the ER to the Golgi apparatus in gently lysed yeast spheroplasts is mediated by diffusible vesicles. These transport vesicles contain core-glycosylated pro-alpha-factor and are physically separable from donor ER and target Golgi compartments. The formation of diffusible vesicles from the ER requires ATP, Sec12p,(More)
Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective(More)
We have isolated vesicles that mediate protein transport from the ER to Golgi membranes in perforated yeast. These vesicles, which form de novo during in vitro incubations, carry lumenal and membrane proteins that include core-glycosylated pro-alpha-factor, Bet1, Sec22, and Bos1, but not ER-resident Kar2 or Sec61 proteins. Thus, lumenal and membrane(More)
The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its(More)
Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils(More)
Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein(More)
All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387-395; Makio, T., L.H. Stanton, C.-C. Lin, D.S.(More)
Bioinformatics of disordered proteins is especially challenging given high mutation rates for homologous proteins and that functionality may not be strongly related to sequence. Here we have performed a novel bioinformatic analysis, based on the spatial clustering of physically relevant features such as binding motifs and charges within disordered proteins,(More)