Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 → K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-␣ and -␤ (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase. In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins. Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost. A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins. These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins. These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZot fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.Protein translocation across the cytoplasmic membrane of Escherichia coli is facilitated by a cytoplasmic chaperone (SecB in many cases), the peripheral membrane ATPase (SecA), and membrane-embedded factors (SecY, SecE, SecD, and SecF) (26,35). Reconstitution studies have established that SecA, SecE, and SecY are essential components for the in vitro translocation reactions (1, 9).SecY spans the membrane 10 times with both of its termini facing the cytoplasm (4). Genetic (7,29) ompT::kan) was constructed by cotransducing secE501 and an adjacent argE::TnlO marker into AD202 (MC4100 ompT::kan) (5) by using P1 vir prepared from PR520 (23). For pulse-chase experiments, cells were grown in M9 medium (31) supplemented with 0.4% glycerol, 2 ,ug of thiamine per ml, and 20 ,ug of each amino acid except methionine and cysteine per ml. Ampicillin (50 ,ug/ml) and/or chloramphenicol (20 jig/ml) was added when plasmid-bearing cells were grown. To maximize expression of genes under lac promoter control, 1 mM cyclic AMP was added.Plasmids. pKY318 was a pBR322-derived secY plasmid; the 2.8-kb PstI fragment of the spc operon on pNO1573 (2) was cloned into pKY225, a lac promoter vector identical to pKY184 (33) except that the ,-lactamase (bla) gene was derived from pUC119 (34). pKY248 was a pACYC184-derived secY plasmid; the HindIII-BamHI fragment of pKY107 containing secY (29) was cloned into pKY238 (29), a lac promoter vector based on pACYC184. pKY258, carrying the secY-lacZa fusion gene, was constructed as follows. The 1.5-kb HindlIl fragment that contained secY from pKY3 (28) was cloned into pKY184. The secY and lacZoa reading frames were then fused by the method of Kunkel et al. (18) by using a synthetic 60-mer oligonucleotide that consisted of the last 10 codons of secY and codons 7 to 16 of lacZot from pUC9 (with a regenerated Hindlll site at the junction), yielding pKY234. Finally, the HindlIl...

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p͞ Crm1p͞exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin͞karyopherin ␤ superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.