RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells slows cell growth. This toxicity depends on the presence of a series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.Proteins and RNAs are continuously moving in and out of the nucleus via the nuclear pore. Proteins destined for the nucleus often contain short stretches of amino acids, termed nuclear localization sequences (NLSs), that can direct them to the nucleus (15,17,34). Other proteins may enter the nucleus by associating with an NLS-bearing protein (38). Once at the pore, the protein to be imported binds and is transported in a reaction that requires ATP (26, 28) and proteins of the nuclear pore complex (12,13,24,32). Much less is known about exit of macromolecules from the nucleus. All classes of RNAs are thought to be exported to the cytoplasm as RNA-protein complexes via the nuclear pores (10). Most likely this is also an active process that may involve specific targeting of macromolecules. Experiments with microinjected RNAs indicate that exit of RNA from the nucleus is an energy-dependent, facilitated, and saturable process (7,37).Some nuclear proteins, in particular those that bind RNA, repeatedly shuttle in and out of the nucleus (9). The functional significance of this export-reimport is still unclear. However, one proposal is that nuclear shuttling proteins may be directly involved in nucleocytoplasmic exchange of macromolecules. By one model, certain RNA-binding proteins would be part of the export substrate that moves with the RNA from the nucleus via the pore. Following release of their RNA "cargo" by exchange with cytoplasmic RNA-binding proteins, the proteins that accompanied RNA out would rapidly reenter the nucleus. Implicit in this model is the idea that some proteins would contain information that not only targets them to the

We have isolated mutants of the yeast Saccharomyces cerevisiae that are defective in localization of nuclear proteins. Chimeric proteins containing the nuclear localization sequence from SV40 large T-antigen fused to the N-terminus of the mitochondrial Fl3-ATPase are localized to the nucleus. Npl (nuclear protein localization) mutants were isolated by their ability to grow on glycerol as a consequence of no longer exclusively targeting SV40-F1f-ATPase to the nucleus. All mutants with defects in localization of nucleolar proteins and histones are temperature sensitive for growth at 36°C. Seven alleles of NPL3 and single alleles of several additional genes were isolated. NPL3 mutants were studied in detail. NPL3 encodes a nuclear protein with an RNA recognition motif and similarities to a family of proteins involved in RNA metabolism. Our genetic analysis indicates that NPL3 is essential for normal cell growth; cells lacking NPL3 are temperature sensitive for growth but do not exhibit a defect in localization of nuclear proteins. Taken together, these results indicate that the mutant forms of Npl3 protein isolated by this procedure are interfering with nuclear protein uptake in a general manner. INTRODUCTIONCertain proteins enter the nucleus by recognition of a nuclear localization sequence (NLS) in the transported protein (Silver, 1991). After NLS-dependent binding either in the cytoplasm or at the nuclear pore complex, proteins are transported through the pore in an ATPdependent manner. This two-step model is based on observations made on the behavior of proteins microin-

The addition of unsaturated fatty acids to cultures of Saccharomyces cerevisiae significantly altered the microsomal lipid composition. Supplementation with either of the naturally occurring palmitoleic (16:1) or oleic (18:1) acids caused increased levels in membrane phospholipids and reduced levels of the complementary acid. Growth in the presence of equimolar quantities of 16:1 and 18:1 acids, however, produced a fatty acid composition similar to that found in unsupplemented cell membranes. Linoleic acid (18:2) was not found in S. cerevisiae grown under normal conditions. It was preferentially internalized and incorporated into microsomes, however, at levels exceeding 50% of the total fatty acid species. This resulted in an almost total loss of 16:1 and a reduction of 18:1 to 25% of its normal level. The A-9 fatty acid desaturase, a microsomal enzyme that forms 16:1 and 18:1 from saturated acyl coenzyme A precursors, was affected by the presence of exogenous fatty acids. Enzyme activity toward the 16:0 coenzyme A substrate was elevated in microsomes from saturated-fattyacid-supplemented cultures and sharply repressed following the addition of unsaturated fatty acids, including 18:2. Northern (RNA blot) and slot-blot analyses of mRNA encoded by the OLE] gene, which appears to be the structural gene for the A-9 desaturase, indicated that it was sharply reduced in unsaturated-fatty-acid-fed cells. These data suggest that a significant part of the regulation involves modulation of available transcripts.Approximately 70% of the fatty acids in membrane lipids of the yeast Saccharomyces cerevisiae consist of palmitoleic acid (16:1) and oleic acid (18:1) (12). The remaining fatty acids are saturated, consisting primarily of palmitic acid (16:0) and lesser amounts of stearic acid (18:0) and myristic acid (14:0). Unlike most other fungi, whose most abundant unsaturated fatty acids are the di-and trienoic linoleic (18:2) and ox-linolenic (18:3) species (27), S. cerevisiae synthesizes only monounsaturated acids when grown under normal laboratory conditions. Unsaturated fatty acids are synthesized in fungal and animal cells by fatty acid desaturases, which are hydrophobic microsomal enzymes (3,4,23). The A-9 desaturase catalyzes the formation of the initial double bond between the 9th and 10th carbons of both palmitoyl (16:0) and stearoyl (18:0) coenzyme A (CoA) substrates to make 16:1 and 18:1. Introduction of the double bond is a complex reaction requiring the removal of electrons and hydrogens from the hydrocarbon chain of the fatty acid and the transfer of an additional two electrons from NADH to molecular oxygen via cytochrome b5 and b5 reductase (4,22).Considerable evidence has accumulated showing that the A-9 fatty acid desaturase in animal cells is regulated by dietary fatty acids. For example, in rat and chicken liver (18,19,25), the enzyme is repressed by dietary unsaturated fatty acids. The desaturase is the only component of the enzyme system that appears to be regulated, however, since cytochrome b5 and b5 red...

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Npl3p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism.

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Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the anti-idiotype antibodies compete for binding of nuclear proteins in vitro. We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.