The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.Retroviruses and long terminal repeat (LTR)-containing retrotransposons possess similar methods of propagation that include the conversion of their mRNA into cDNA by reverse transcriptase (RT) and the insertion of this double-stranded DNA into the host genome by integrase (IN). Because reverse transcription occurs in the cytoplasm, the preintegration complexes (PIC) of cDNA and IN must be transported into the nucleus for integration to occur. Nuclear pore complexes (NPC) are assemblies of more than 50 proteins that provide the means for selective passage of proteins and nucleic acids between the cytoplasm and the nucleus (55, 58). Although significant progress has been made describing the families of transport factors that deliver nuclear localization sequence (NLS)-containing proteins to the nucleus (13,21,51,53,65), the current models of nuclear import do not directly address how macromolecules as large as virus complexes pass through the nuclear pore.The length and diameter of the transport channel have been measured by testing nucleoplasmin-coated gold particles of various sizes for the ability to pass through the nuclear pore. The maximum diameter of the channel was found to be 20 to 25 nm, and this passage extends approximately 50 nm across the nuclear envelope (15, 46). Large macromolecular substrates that must in some form pass through the NPCs in intact nuclear envelopes include the 50-nm virus particles of simian virus 40 (SV40) (24, 49, 70), the 90-nm particles of adenovirus (24, 60), and the 160S PIC of human immunodeficiency virus (HIV). Although the adenovirus particles attach to the NPC and disassemble before the transport of the DNA-protein VII complex, SV40 appears to pass through the NPC as virion particles (25,49,70). The nuclear import of the HIV PIC reflects not only the presence of NLS activ...

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (i) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.Like many other microorganisms, Saccharomyces cerevisiae can utilize ammonium as a sole source of nitrogen. In baker's yeast, utilization of ammonia occurs exclusively via its incorporation into glutamate and glutamine (37). This process occurs in two ways. The first is a combination of two successive reactions catalyzed by the glutamine synthetase (16, 41) and the glutamate synthase (52), respectively. This pathway is of minor importance, as shown by the fact that mutants lacking glutamate synthase activity grow as well as wild-type cells in medium containing ammonium sulfate as the sole nitrogen source (39). The second pathway, which combines two reactions catalyzed, respectively, by NADP-linked glutamate dehydrogenase (NADP-GDH, product of the GDH1 gene) (42, 44) and glutamine synthetase, constitutes the major pathway for the assimilation of ammonia. In ammonium sulfate medium, mutants lacking NADP-GDH activity grow at only about half the rate of wild-type cells (25).The reductive amination of ␣-ketoglutarate by NADP-GDH is not only a key step for ammonia utilization but also an important connection point between carbon metabolism and nitrogen metabolism. For these reasons, regulation of GDH1 gene expression is an important and probably complex issue. Activation of GDH1 expression by the transcription factor Leu3p has been recently repor...

Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein.