Abstract. We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein, p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSRI protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSRI (p67) is required for normal cell growth.

Nuclear proteins contain specific regions that are required for entry into the nucleus. Using ligand blotting, we have shown that a 67-kDa yeast nuclear envelope protein (p67) recognizes synthetic peptides containing the yeast histone H2B or simian virus 40 large tumor antigen nuclear localization sequence. Both free peptide and peptide conjugated to human serum albumin are recognized. The interaction between p67 and the nuclear localization sequences is specific; neither a mutant peptide that is incompetent for nuclear transport in vivo nor HSA can interact with p67 on blots. Moreover, although the wild-type peptide competes for binding to p67, the mutant peptides do not. p67 appears to be located at the nuclear envelope and is not present in other subcellular fractions. The nuclear localization sequence-binding protein is not extracted from the nuclear envelope with nonionic detergents and only partially extracted with high-salt buffer or 8 M urea, suggestive of a tight association with the nuclear envelope. Together our results are consistent with a role for p67 in nuclear transport.Studies using dextrans and small nonnuclear proteins have shown that the nuclear envelope is a molecular sieve for proteins the size of 20-40 kDa (1). Evidence suggests that nuclear proteins larger than 40 kDa are actively transported into the nucleus by way of the nuclear pores (see reviews in refs. 2 and 3). The transport of nuclear proteins has been shown to occur in two steps: binding and subsequent translocation. Only the translocation step requires ATP (4-7). Endogenous nuclear localization sequences have been shown to direct nuclear proteins to the nucleus and, through gene fusions, can also mediate the transport of cytoplasmic and bacterial proteins into the nucleus (see reviews in refs. 2 and 3). In addition, synthetic peptides containing the nuclear localization sequence of simian virus 40 large tumor antigen (SV40 T antigen), when chemically conjugated to nonnuclear proteins, are also capable of targeting them to the nuclear compartment (8-10).Recently, two different investigators have used chemical cross-linking methods in rat liver to show that proteins in the cytoplasm and at the nuclear envelope specifically interact with synthetic peptides containing nuclear localization sequences. These are the first candidates for proteins likely to be involved in the initial signal-recognition step required for nuclear transport. Using a synthetic SV40 T-antigen peptide, Adam et al. (11) found a major 60-kDa and a minor 70-kDa protein that were distributed primarily in the cytoplasm but also in the nuclear interior and nuclear envelope. The authors suggest a multistep model for nuclear protein transport in which a cytoplasmic receptor binds to a nuclear protein and carries the protein into the nucleus via a second receptor at the nuclear envelope. Prompt dissociation within the nucleus would then lead to recycling of the receptor to the cytoplasm.Yamasaki et al. (12) also reported additional proteins that recognize a num...

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.