The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants.

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-alpha export.

Proteins bearing canonical nuclear localization sequences are imported into the nucleus by the importin/karyopherin-alpha/beta heterodimer. Recycling of the importin-alpha subunit to the cytoplasm requires the action of Cas, a member of the importin-beta superfamily. In the yeast Saccharomyces ceresivisiae, the essential gene CSE1 encodes a Cas homologue that exports the yeast importin-alpha protein Srp1p/Kap60p from the nucleus. In this report, we describe a role for the FXFG nucleoporin Nup2p, and possibly the related Nup1p, in the Cse1p-mediated nuclear export pathway. Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 accumulate Srp1p in the nucleus. Similarly, Cse1p is displaced from the nuclear rim to the nuclear interior in deltanup2 cells. We do not observe any biochemical interaction between Cse1p and Nup2p. Instead, we find that Nup2p binds directly to Srp1p. We have localized Nup2p to the interior face of the nuclear pore complex, and have shown that its N terminus is sufficient for targeting Nup2p to the pore, as well as for binding to Srp1p. Taken together, these data suggest that Nup2p is an important NPC docking site in the Srp1p export pathway.

SAC3 may link nuclear protein export to cell cycle progression.

Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996) J. Cell Sci. 109, 1575-1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of SAC3 function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that SAC3 interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.

In or out? Regulating nuclear transport.

The compartmentalization of proteins within the nucleus or cytoplasm of a eukaryotic cell offers opportunity for regulation of cell cycle progression and signalling pathways. Nuclear localization of proteins is determined by their ability to interact with specific nuclear import and export factors. In the past year, substrate phosphorylation has emerged as a common mechanism for controlling this interaction.

Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae.

In metazoan cells, the CAS protein has been shown to function as a recycling factor for the importin-alpha subunit of the classical nuclear localization signal receptor, exporting importin-alpha from the nucleus to allow its participation in multiple rounds of nuclear import. CAS is a member of a family of proteins that bear homology to the larger subunit of the nuclear localization signal receptor, importin-beta, and that are found in all eukaryotes from yeast to humans. Sequence similarity identifies the product of the Saccharomyces cerevisiae CSE1 gene as a potential CAS homologue. Here we present evidence that Cse1p is the functional homologue of CAS: Cse1p is required to prevent accumulation of Srp1p/importin-alpha in the nucleus, it localizes to the nuclear envelope in a pattern typical of nuclear transport receptors, and it associates in vivo with Srp1p in a nucleotide-specific manner. We show further that mutations in CSE1 and SRP1 have specific effects on their association and on the intracellular localization of Cse1p.

Furan fatty acids determined as oxidation products of conjugated octadecadienoic acid.

The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50 degrees C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.