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.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called ‘der’ for ‘degradation in the ER’. DER1 was cloned by complementation of the der1–2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1‐deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

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-␤-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.In eukaryotic cells, all transport between the nuclear interior and the cytoplasm occurs through the nuclear pore complexes (NPCs) (reviewed in reference 16). According to the data that have accumulated during the last few years, proteins destined to enter the nucleus associate in the cytoplasm with receptors that recognize and bind specific sequences, termed nuclear localization signals (NLSs). These complexes are targeted to the NPC and are translocated into the nucleoplasm, where the import cargo is released and the receptor is recycled to the cytoplasm (reviewed in references 13, 31, 33, 65, and 68). In the case of the basic-type (classical) NLS, the receptor consists of importin ␣ (karyopherin ␣), the NLS-binding component, and importin ␤ (karyopherin ␤), which can interact with repeatcontaining nucleoporins and is responsible for docking to the NPC. Importin ␤ belongs to a large protein family whose members are characterized by the presence of an amino-terminally located Ran-GTP binding domain (23,32). Other members of this family include transportin and Kap123p (Yrb4p), which respectively directly bind to some hnRNP proteins and ribosomal proteins, and mediate their nuclear import (24,72,79,83,96). Similar functions have also been proposed for their homologues Kap104p (1) and karyopherin ␤3 (105). Recently two more importin ␤ homologues, Mtr10p and Sxm1p, have been shown to function as import receptors for Npl3p (a yeast hnRNP protein) and Lhp1p (the yeast La homologue), respectively (71,78,86).The principles of active nuclear protein import may also apply to active nuclear export of proteins and RNA. Indeed, two members of the importin ␤ family have been shown to be involved in nuclear export processes and were therefore termed exportins (reviewed in reference 102). Export of importin ␣ from the nucleus is mediated by CAS (57), while CRM1 functions as an export receptor for the leucine-rich nuclear export signal (NES) (22,26,56,67,69,98). This type of NES can mediate n...

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cellpermeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.chemical biology ͉ growth factor signaling ͉ phosphatase inhibition ͉ virtual drug screening

Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. 1998 John Wiley & Sons, Ltd.