Cselp functions as the nuclear export receptor for importin α in yeast

Künzler, Markus; Hurt, Eduard C.

doi:10.1016/s0014-5793(98)00892-8

Cited by 83 publications

(77 citation statements)

References 70 publications

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“…2A). This is con-sistent with results showing that Gsp1p, Cse1p, and Kap60p form an exportin-cargo complex (13)(14)(15), which presumably translocates across the NPC by interacting with FG Nups.…”

Section: Identification Of Nup159p-and Cse1p-interacting Proteinssupporting

confidence: 79%

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Allen

Patel

Huang

et al. 2002

Molecular & Cellular Proteomics

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The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA.More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K D ‫؍‬ 1.5 M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

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Section: Identification Of Nup159p-and Cse1p-interacting Proteinssupporting

confidence: 79%

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Allen

Patel

Huang

et al. 2002

Molecular & Cellular Proteomics

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“…Importin ␤ was expressed and purified as described previously (26). GSTimportin ␣ and GST-A3-importin ␣ were expressed as described elsewhere (40).…”

Section: Methodsmentioning

confidence: 99%

“…Our in vitro data showing that the A3 mutations do not appear to have a direct impact on the binding of Cse1p to importin ␣ suggest that this defect is due to impaired release of cargo in the nucleus. Previous work has shown that the Cse1p/CAS association with importin ␣ does not depend on the N-terminal domain (40,48). In fact, the Cse1p/ CAS binding site has been mapped to the C terminus of human importin ␣ (21).…”

Section: Table II Binding Of Importin ␣ Proteins To Nls-cargomentioning

confidence: 96%

The Auto-inhibitory Function of Importin α Is Essentialin Vivo

Harreman

Hodel²,

Fanara³

et al. 2003

Journal of Biological Chemistry

104

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Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/ karyopherin ␣ and ␤. The importin ␣ subunit recognizes classical NLS sequences, and the importin ␤ subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin ␤ binding (IBB) domain of importin ␣ regulates NLS-cargo binding in the absence of importin ␤ in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin ␣ protein. These mutants dissect the two functions of the N-terminal IBB domain, importin ␤ binding and autoinhibition. One of these importin ␣ mutations, A3, decreases auto-inhibitory function without impacting binding to importin ␤ or the importin ␣ export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin ␣ remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin ␣ is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin ␣ to the cytoplasm.In eukaryotes, the nuclear envelope provides an essential barrier that separates the nuclear genome from the intermediary metabolism, signaling systems, and translation machinery of the cytoplasm. Selective bi-directional transport of macromolecules across this nuclear envelope regulates critical cellular processes such as gene expression (1, 2). All nucleocytoplasmic transport of macromolecules occurs through large proteinaceous structures, called nuclear pore complexes (NPC), 1 that perforate the nuclear envelope (3, 4). These macromolecular cargoes are specifically targeted to and transported through NPCs by a family of soluble nuclear transport receptors (5, 6).The small GTPase, Ran, governs the interactions between the nuclear transport receptors and macromolecular cargoes (5, 7). Import receptors bind cargo in the absence of RanGTP, whereas export receptors bind cargo in a trimeric complex with RanGTP (5,8). This mode of regulation requires an asymmetric distribution of RanGTP, with more RanGTP in the nucleus than in the cytoplasm. To achieve this asymmetry, the Ran regulatory proteins are compartmentalized with the GTPase activating protein (RanGAP), which generates RanGDP, in the cytoplasm (9) and the guanine nucleotide exchange factor (RCC1), which generates RanGTP, in the nucleus (10).The best-characterized nuclear import process occurs via receptor recognition of a classical nuclear localization signal (NLS). This classical NLS is typified by a cluster of basic amino acids (monopartite) or two clusters of basic amino acids separated by a 10 -12 amino acid linker (bipartite) (11, 12). A heterodimeric import receptor, composed of importins ␣ and ␤ (also known as karyopherin ␣ and ␤), mediates the nuclear import of proteins that contain a classical NLS (13-15). Over the last several years many...

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“…We explored the possibility that CSE1 and SMC4 could play roles in regulating DNA replication. CSE1 is responsible for the nuclear shuttling of the nuclear transporter importin ␣ (Hood and Silver, 1998;Kunzler and Hurt, 1998;Solsbacher et al, 1998) and a role for CSE1 in mitosis (but not in S phase) has been described (Xiao et al, 1993;Schroeder et al, 1999). We synchronized cells in G1, released them into the cell cycle after shut-off of TetO 7 -CSE1, and measured the DNA contents of the cells by flow cytometry ( Figure 6A).…”

Section: S Phasementioning

confidence: 99%

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Peña‐Castillo

Mnaimneh

et al. 2006

MBoC

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Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO 7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. INTRODUCTIONCell division is a fundamental biological process that in all organisms consists of a series of closely coordinated events. In the budding yeast Saccharomyces cerevisiae, the cell division cycle begins with an initial growth phase, G1, during which time the cell increases in mass and volume. The transition from G1 into S phase is marked by progression through Start (the point of commitment to cell division), initiation of nuclear DNA synthesis, and the emergence of a bud that will form the new daughter cell. S phase is followed by a second growth phase, G2, which is in turn followed by nuclear division, and then cell separation. A very similar series of events occurs in all eukaryotes, with the obvious exception of bud emergence. Because of the fundamental biological importance of cell cycle progression and its importance in both human development and diseases such as cancer, identification of the molecular determinants of specific stages of the eukaryotic cell cycle has been a subject of intense study for several decades.The morphological landmarks of the cell cycle stages in budding yeast, most notably the size of the bud relative to the size of the mother cell, allows the identification of mutants blocked at specific stages of the cell cycle and thereby forms the basis for the classic cell cycle screens (Hartwell et al., 1970;Culotti and Hartwell, 1971;Hartwell, 1971aHartwell, , 1971bHartwell, , 1973Moir et al., 1982). These genetic screens, using conditional temperature-sensitive mutants, identified more than 50 genes that are required for specific stages in the cell division cycle and so were termed CDC genes. On average 4.6 alleles were identified for each CDC gene in the original screens, suggesting the number of cdc mutan...

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Cselp functions as the nuclear export receptor for importin α in yeast

Cited by 83 publications

References 70 publications

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

The Auto-inhibitory Function of Importin α Is Essentialin Vivo

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

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