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-␤-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...

Yeast Nuclear Envelope Subdomains with Distinct Abilities to Resist Membrane Expansion

“…pASZ11-ADE2-PUS1-GFP, encoding PUS1-GFP (Hellmuth et al, 1998), and pASZ11-ADE2-NUP49-GFP, encoding NUP49-GFP (Belgareh and Doye, 1997), were gifts from Ed Hurt (University of Heidelberg, Heidelberg, Germany). These plasmids were modified by PCR-mediated replacement of the ADE2 gene with the URA3 gene to yield plasmids pPUS1-URA and pNUP49-URA, respectively.…”

Have tRNA, will travel

“…Coupling of splicing and export of tRNA could also conveniently explain the role of two proteins, Rna1 and Los1, mutations in which resulted in the accumulation of unspliced tRNA (10,11). Rna1 protein was shown to be the GTPase activating protein (GAP) of the Ran cycle (12, 13), and Los1 protein was shown to be a tRNA exportin (14)(15)(16).…”

Conformational Variability of Nucleo-cytoplasmic Transport Factors

“…In the case of nuclear export, several mediators have been studied to date. They include the importin ␣ export factors (human CAS and yeast Cse1) (10 -12), tRNA export factors (human Xpo-t and yeast Los1) (13)(14)(15), and NES 1 export factors (human Crm1 and yeast Xpo1) (16 -19). The transport machinery is remarkably versatile, with individual karyopherins recognizing multiple cargoes and with individual cargoes binding to multiple karyopherins (see reviews, Refs.…”