George Simos

Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores.

ABSTRACT We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating eitherMEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.

Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA

ABSTRACT Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-β-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

THE YEAST PROTEIN ARC1P BINDS TO TRNA AND FUNCTIONS AS A COFACTOR FOR THE METHIONYL- AND GLUTAMYL-TRNA SYNTHETASES

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore‐associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl‐tRNA and glutamyl‐tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1‐ mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N‐terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV‐cross‐linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl‐tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

Mtr10p functions as a nuclear import receptor for the mRNA‐binding protein Npl3p

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)‐tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA‐binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)‐containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non‐repetitive C‐terminal tail. Mtr10p interacts in vitro with Gsp1p‐GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran‐GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran‐GTP and newly synthesized mRNA.

Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1α, involves several post-translational modifications. To study HIF-1α phosphorylation, we have used human full-length recombinant HIF-1α as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1α. Analysis of in vitro phosphorylated HIF-1α by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1α. When these mutant forms of HIF-1α were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1α mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1α. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1α mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1α. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1α by blocking its CRM1-dependent nuclear export.

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide‐specific and intron‐dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre‐tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(‐) pus1(‐) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

THE LAMIN B RECEPTOR (LBR) PROVIDES ESSENTIAL CHROMATIN DOCKING SITES AT THE NUCLEAR ENVELOPE

Morphological studies have established that peripheral heterochromatin is closely associated with the nuclear envelope. The tight coupling of the two structures has been attributed to nuclear lamins and lamin‐associated proteins; however, it remains to be determined which of these elements are essential and which play an auxiliary role in nuclear envelope‐chromatin interactions. To address this question, we have used as a model system in vitro reconstituted vesicles assembled from octyl glucoside‐solubilized nuclear envelopes. Comparing the chromosome binding properties of normal, immunodepleted and chemically extracted vesicles, we have arrived at the conclusion that the principal chromatin anchorage site at the nuclear envelope is the lamin B receptor (LBR), a ubiquitous integral protein of the inner nuclear membrane. Consistent with this interpretation, purified LBR binds directly to chromatin fragments and decorates the surface of chromosomes in a distinctive banding pattern.

A Conserved Domain within Arc1p Delivers tRNA to Aminoacyl-tRNA Synthetases

Two yeast enzymes that catalyze aminoacylation of tRNAs, MetRS and GluRS, form a complex with the protein Arc1p. We show here that association of Arc1p with MetRS and GluRS is required in vivo for effective recruitment of the corresponding cognate tRNAs within this complex. Arc1p is linked to MetRS and GluRS through its amino-terminal domain, while its middle and carboxy-terminal parts comprise a novel tRNA-binding domain. This results in high affinity binding of cognate tRNAs and increased aminoacylation efficiency. These findings suggest that Arc1p operates as a mobile, trans-acting tRNA-binding synthetase domain and provide new insight into the role of eukaryotic multimeric synthetase complexes.

Lamins and lamin-associated proteins

A variety of morphological and biochemical studies have established that the nuclear lamins play an important role in nuclear structure and dynamics. Recent work reveals the existence of specialized lamin isotypes and novel pathways of modulation of lamin import into the nucleus via phosphorylation by protein kinase C. Other studies also unveil a wide spectrum of molecular interactions between the lamin proteins and integral membrane components of the nuclear envelope.

The lamin B receptor-associated protein p34 shares sequence homology and antigenic determinants with the splicing factor 2-associated protein p32.

The lamin B receptor (p58) is an inner nuclear membrane protein that forms an in vivo complex with the nuclear lamins, a nuclear envelope kinase, and two other nuclear proteins with apparentM r of 18,000 (p18) and 34,000 (p34). We now report the isolation of p34 by partial dissociation of the immunoaffinity‐purified p58 protein complex. Determination of the N‐terminal amino acid sequence of purified p34 shows that this polypeptide is homologous to p32, a splicing factor 2 (SF2)‐associated protein. The relatedness between p34 and p32 can be further established by showing that antibodies raised against N‐ and C‐terminal peptides of p32 cross‐react with purified p34. As the amino acid sequence of p58 contains an arginine/serine (RS)‐rich region similar to the RS‐rich region found in SF 2, we speculate that these domains provide binding sites for p34 and that this protein may be a linker between the nuclear membrane and intranuclear spliceosomal substructures.