Ran/TC4, a Ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control of protein movement into the nucleus cytoplasmic accumulation mRNA. Saccharomyces cerevisiae contains two homologues mammalian encoded by GSP1 GSP2 genes. We constructed yeast strains that overproduce either wild-type Gsp1 or form with glycine-21 converted to valine (Gsp1-G21V), which we show stabilizes GTP-bound form. Cells producing Gsp1-G21V defects localization nuclear proteins;...

The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. same result is obtained when examined living using fused naturally green fluorescent These findings suggest role for Rna1p trafficking proteins across membrane. To investigate this more directly, an vitro assay...

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes Saccharomyces cerevisiae homolog vertebrate receptor importin. Importin was isolated as factor required for initial targeting step substrate to envelope mammalian vitro assay. We show that yeast Srp1 similarly protein import. In addition, also execution mitosis: demonstrate cells containing SRP1 arrest with G2/M phenotype manner analogous classic cdc...

Eukaryotic cells contain multiple Hsp70 proteins and DnaJ homologues. The partnership between a given its interacting could, in principle, be determined by their cellular colocalization or specific protein-protein interactions. yeast SCJ1 gene encodes one of several homologues the bacterial chaperone DnaJ. We show that Scj1p is located lumen endoplasmic reticulum (ER), where it can function with Kar2p (the ER-lumenal BiP/Hsp70 yeast). region common to all (termed J domain) from swapped for...

Radiolabeled preprocecropin B, with an alpha-amidated COOH terminus, and A, extended at the terminus by a glycine residue, were synthesized solid-phase methods. The respective syntheses interrupted intervals to allow preparation of predicted procecropins A B as well three other truncated derivatives cecropin precursors. All synthetic peptides purified near homogeneity reverse-phase liquid chromatography their purity was established analytical high performance chromatography, gel...

Within a single generation time growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone are targeted to assembly site in nucleolus. Here, we report discovery of conserved nuclear carrier Tsr2 that coordinates transfer r-protein eS26 earliest assembling pre-ribosome, 90S. In vitro studies revealed efficiently dissociates importin:eS26 complexes via an...

Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by heterodimeric transporter importin. Importin α binds NLS and importin β, which carries it through pore complex (NPC). disassembles in nucleus, evidently binding of RanGTP β. The subunits exported separately. We investigated role Cse1p, Saccharomyces cerevisiae homologue human CAS, export Srp1p (yeast α). Cse1p is located predominantly but also present cytoplasm at NPC. analyzed vivo fused green fluorescent...

In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that embedded in nuclear envelope via its central plaque, whereas outer and inner plaques exposed to cytoplasm nucleoplasm, respectively. How assembles poorly understood. We show inner/central plaque composed of stable subcomplex, containing γ-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, Spc29p. Spc29p acts as linker between component Spc42p Spc110p. Evidence...

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability translocate purified vesicles on depends presence an accessory complex known as dynactin. We have cloned and characterized a novel gene,NIP100, which encodes yeast homologue dynactin protein p150 glued . Like strains lacking heavy chain Dyn1p or centractin Act5p, nip100Δ are viable but undergo significant number failed mitoses mitotic spindle does not properly partition into daughter cell....

Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires ribonucleoparticles, signal recognition particle, and ribosome is tightly coupled to ongoing protein synthesis. We have shown previously that small can translocate post-translationally in a reaction does not require these ribonucleoparticles. now report one protein, synthetic hybrid between preprocecropin A dihydrofolate reductase, translocates both cotranslationally (with aid particle ribosome)...

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by cofactors that contain domains homologous to bacterial DnaJ. Of three DnaJ homologues in yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related DnaJ, hence it a probable cofactor for Kar2p, major Hsp70 RER. However, physiological role has remained obscure due lack an obvious defect Kar2p-mediated pathways scj1 null mutants. Here, we show Δscj1 mutant hypersensitive...

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and beta. Srp1p, Saccharomyces cerevisiae homologue alpha, returns from in complex with its export factor Cse1p Gsp1p (yeast Ran) GTP-bound state. We studied role nucleoporin Nup2p transport cycle Srp1p. Cells lacking NUP2 show specific defect both NLS import Srp1p export, indicating that required for efficient bidirectional across pore (NPC). located at side central...

We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The uses cells that been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show the envelope remains intact but plasma membrane is perforated. In presence of ATP cytosol derived or mammalian cells, a containing localization sequence (NLS) SV40 large T-antigen transported into nucleus. Proteins with mutant NLSs are not imported....

The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has well defined role in cell cycle-specific transcription. serves second function the transcriptional response to wall stress which activated Mpk1 mitogen-activated protein kinase integrity signaling pathway forms complex with Swi4, DNA binding subunit SBF, conferring upon ability bind activate FKS2. Although Mpk1–Swi4 formation activation FKS2 does not require catalytic activity, phosphorylated by must be...

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content potential coiled-coil structure similar to myosin. Nuf2 is associated spindle pole body (SPB) as determined by coimmunofluorescence known SPB proteins. appears be localized intranuclear region and candidate for involved in separation. nuclear association can disrupted, part, 1 M salt but not detergent Triton X-100. All removed from nuclei 8 urea extraction. In this regard, other...

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich signal (NES). Xpo1p, NES-containing protein, and GTP-bound Ran form complex in nucleus that translocates across pore. We have identified Yrb1p as major Xpo1p-binding protein Saccharomyces cerevisiae extracts presence of Gsp1p (yeast Ran). cytoplasmic at steady-state but shuttles continuously between cytoplasm nucleus. Nuclear import mediated by two separate targeting signals. Export from requires does not...

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), required for proper NPC distribution and assembly. To characterize NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a novel partner, import factor Kap123. In vitro revealed direct interaction between C-terminal domain (CTD) silico analysis predicted presence two amphipathic α-helices within Pom33-CTD. Circular dichroism liposome...