The arrest of secretion response in yeast: signaling from the secretory path to the nucleus via Wsc proteins and Pkc1p.

The arrest of secretion response (ASR) in sec mutants reversibly inhibits nuclear import and relocates nuclear proteins to the cytoplasm. sec mutants also relocate nucleoporins; however, endocytic and Golgi-to-vacuole transport mutants do not cause relocation. The ASR requires Wsc membrane proteins that are trapped along the secretory path, rather than those which are at the plasma membrane. The activity of the downstream kinase, Pkc1p, is also required; however, the Pkc1p MAP kinase cascade is not. sec mutants initiate compensatory transcriptional changes distinct from those of the unfolded protein response.

Nuclear relocation of normal huntingtin.

In Huntington's Disease (HD), the huntingtin protein (Htt) includes an expanded polyglutamine domain. Since mutant Htt concentrates in the nucleus of affected neurons, we have inquired whether normal Htt (Q16--23) is also able to access the nucleus. We observe that a major pool of normal full-length Htt of HeLa cells is anchored to endosomes and also detect RNase-sensitive nuclear foci which include a 70-kDa N-terminal Htt fragment. Agents which damage DNA trigger caspase-3-dependent cleavage of Htt and dramatically relocate the 70 kDa fragment to the nucleoplasm. Considering that polyglutamine tracts stimulate caspase activation, mutant Htt is therefore poised to enter the nucleus. These considerations help rationalize the nuclear accumulation of Htt which is characteristic of HD and provide a first example of involvement of caspase cleavage in release of membrane-bound proteins which subsequently enter the nucleus.

The Saccharomyces cerevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-A0 fragment of 35S pre-rRNA, both of which are carried out by the exosome.

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

Perturbation of the nucleus: a novel Hog1p-independent, Pkc1p-dependent consequence of hypertonic shock in yeast.

Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the "cell integrity" MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.

Control of nuclear export of hnRNP A1.

mRNA export is mediated by RNA-binding proteins which shuttle between the nucleus and cytoplasm. Using an in vitro unidirectional export assay, we observe that the shuttling mRNA-binding protein, hnRNP A1, is exported only extremely slowly unless incubations are supplemented with snRNA-specific oligonucleotides which inhibit splicing. In vivo microinjection experiments support this conclusion. Like many examples of nucleocytoplasmic transport, export of hnRNP A1 requires energy and is sensitive to the presence of wheat germ agglutinin. It does not, however, require supplementation with cytoplasmic proteins. Although the exportin, Crm1, is needed for export of several varieties of RNA, both the in vitro assay and in vivo assays show that it is not required for export of hnRNP A1. In vitro and in vivo studies also show that inhibition of transcription allows continued shuttling of hnRNP A1 and in fact accelerates its export. Judging from the stimulatory effects of targeted destruction of snRNAs, this is likely to reflect completion of the covalent maturation of the RNAs with which hnRNP A1 associates. These observations therefore provide a simple explanation of why multiple RNA-binding proteins relocate to the cytoplasm upon inhibition of transcription in vivo.

Review: dynamic stability of the interphase nucleus in health and disease.

Ongoing export of newly synthesized RNAs, as well as control of transcriptional activity, involves dynamic nucleocytoplasmic transport of proteins. Some proteins that shuttle reside primarily in the nucleus while others are concentrated in the cytoplasm. Moreover, some proteins shuttle continuously, while others shuttle only once. A third group is stimulated to relocate either into or out of the nucleus as a result of interruption of shuttling. In addition to these protein-specific events, several physiological stimuli have global effects on nucleocytoplasmic transport. In related events, selected proteins move between distinct sites in the nucleoplasm, others enter and leave the nucleolus, and still others transit between the nuclear envelope and cytoplasmic membranes. These multiple dynamic distributions provide numerous opportunities for precise communication between spatially distant sites in the cell.

An unexpected link between the secretory path and the organization of the nucleus.

Yeast sec mutations define the machinery of vesicular traffic. Surprisingly, many of these mutations also inhibit ribosome biogenesis by reducing transcription of rRNA and genes encoding ribosomal proteins. We observe that these mutants reversibly inhibit protein import into the nucleus, with import cargo accumulating at the nucleoplasmic face of nuclear pore complexes, as when Ran-GTP cannot bind importins. They also rapidly and reversibly relocate multiple nucleolar and nucleoplasmic proteins to the cytoplasm. The import block and relocation are antagonized by overexpression of yeast Ran, Hog1p kinase, or Ssa/Hsp70 proteins or by inhibition of protein synthesis. These nucleocytoplasmic signaling events document an extraordinary plasticity of nuclear organization.

A Crm1p-independent nuclear export path for the mRNA-associated protein, Npl3p/Mtr13p.

mRNA export involves association of mRNAs with nucleoplasmic proteins, delivery to the nuclear pore complex, translocation to the cytoplasm, and reimport of recycling components. Many yeast mutants inhibit mRNA export, but there is little information concerning the RNA carriers and steps of transport that they affect. The hnRNP/serine-arginine-rich-like protein, Npl3p/Mtr13p, binds poly(A)+ RNA and shuttles between the nucleus and cytoplasm. Its export accelerates on inhibition of RNA synthesis. In vivo tests show that its export requires two proteins with putative leucine-rich nuclear export signals: Gle1p, Mex67p, and several additional nuclear and nuclear pore complex-associated proteins. Surprisingly, a nonnuclear pool of an import factor (the importin alpha homologue, Srp1p) is also required. Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution. A crm1 mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export. Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo. Thus, Npl3p export requires only a subset of proteins implicated in mRNA export, suggesting that more than one mRNA export path exists. A distinct group of mutants, including a mutation of a member of the importin beta superfamily, inhibits Npl3p reimport from the cytoplasm.

Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export.

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Heat shock disassembles the nucleolus and inhibits nuclear protein import and poly(A)+ RNA export.

Heat shock causes major positive and negative changes in gene expression, drastically alters the appearance of the nucleolus and inhibits rRNA synthesis. We here show that it causes many yeast nucleolar proteins, including the fibrillarin homolog Nop1p, to relocate to the cytoplasm. Relocation depends on several proteins implicated in mRNA transport (Mtrps) and is reversible. Two observations indicate, surprisingly, that disassembly results from a reduction in Ssa protein (Hsp70) levels: (i) selective depletion of Ssa1p leads to disassembly of the nucleolus; (ii) preincubation at 37 degrees C protects the nucleolus against disassembly by heat shock, unless expression of Ssa proteins is specifically inhibited. We observed that heat shock or reduction of Ssa1p levels inhibits protein import into the nucleus and therefore we propose that inhibition of import leads to disassembly of the nucleolus. These observations provide a simple explanation of the effects of heat shock on the anatomy of the nucleolus and rRNA transcription. They also extend understanding of the path of nuclear export. Since a number of nucleoplasmic proteins also relocate upon heat shock, these observations can provide a general mechanism for regulation of gene expression. Relocation of the hnRNP-like protein Mtr13p (= Npl3p, Nop3p), explains the heat shock sensitivity of export of average poly(A)+ RNA. Strikingly, Hsp mRNA export appears not to be affected.

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA.

An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm. Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition. There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores. To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C. Several of the corresponding genes have been cloned. Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus. Corresponding dense material is also seen by electron microscopy. MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa. Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA. It therefore may well affect RNA conformation. It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p. Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified. Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export.

Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution.

The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.

A novel class of cell surface glycolipids of mammalian cells. Free glycosyl phosphatidylinositols.

Glycosyl phosphatidylinositol (GPI) lipids function as anchors of membrane proteins, and free GPI units serve as intermediates along the path of GPI-anchor biosynthesis. By using in vivo cell surface biotinylation, we show that free GPIs: 1) can exit the rough endoplasmic reticulum and are present on the surface of a murine EL-4 T-lymphoma and a human carcinoma cell (HeLa), 2) arrive at the cell surface in a time and temperature-dependent fashion, and 3) are built on a base-labile glycerol backbone, unlike GPI anchors of surface proteins of the same cells. The free GPIs described in this study may serve as a source of hormone-sensitive phosphoinositol glycans. The absence of free GPIs from the cell surface may also account for the growth advantage of blood cells in paroxysmal nocturnal hemoglobinuria.

Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae.

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.

Adenoviral E1B-55kDa protein inhibits yeast mRNA export and perturbs nuclear structure.

The mechanisms of export of RNA from the nucleus are poorly understood; however, several viral proteins modulate nucleocytoplasmic transport of mRNA. Among these are the adenoviral proteins E1B-55kDa and E4-34kDa. Late in infection, these proteins inhibit export of host transcripts and promote export of viral mRNA. To investigate the mechanism by which these proteins act, we have expressed them in Saccharomyces cerevisiae. Overexpression of either or both proteins has no obvious effect on cell growth. By contrast, overexpression of E1B-55kDa bearing a nuclear localization signal (NLS) dramatically inhibits cell growth. In this situation, the NLS-E1B-55kDa protein is localized to the nuclear periphery, fibrous material is seen in the nucleoplasm, and poly(A)+ RNA accumulates in the nucleus. Simultaneous overexpression of E4-34kDa bearing or lacking an NLS does not modify these effects. We discuss the mechanisms of selective mRNA transport.

mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

Nucleocytoplasmic transport of mRNA is vital to gene expression and may prove to be key to its regulation. Genetic approaches in Saccharomyces cerevisiae have led to the identification of conditional mutants defective in mRNA transport. Mutations in approximately two dozen genes result in accumulation of transcripts, trapped at various sites in the nucleus, as detected by in situ hybridization. Phenotypic and molecular analyses of many of these mRNA transport mutants suggest that, in yeast, the function of the nucleus is not limited to the biogenesis of pre-ribosomes but may also be important for transport of poly(A)+ RNA. A similar function of the animal cell nucleolus is suggested by several observations.

The nuclear GTPase cycle: promoting peripheralization?

Numerous Ras-like GTPases function as molecular switches in the cytoplasm, but only one has been identified in the nucleus. This nuclear GTPase and its homologues are known in both yeasts and higher organisms and in all cases they are regulated by guanine-nucleotide-exchange factors. The 'nuclear GTPase cycle' created by these components is implicated in mRNA transport from and protein import to the nucleus, as well as in DNA replication, RNA processing and the regulation of the cell cycle. In this article, Alan Tartakoff and Roger Schneiter propose that this GTPase cycle regulates dispersive functions in the nucleoplasm, an idea that explains many of the observed effects of disrupting the cycle.

A yeast protein that bidirectionally affects nucleocytoplasmic transport.

We have identified a temperature-sensitive mutant of Saccharomyces cerevisiae (npl3) that accumulates polyadenylated RNA in the nucleus at 37 degrees C, as judged by in situ hybridization. The strong nuclear signal is not simply due to increased cytoplasmic turnover of mRNA, as reincubation at 37 degrees C with an RNA polymerase inhibitor shows no diminution in the in situ signal. Over several hours at 37 degrees C, the average poly(A) tail length increases and a characteristic ultrastructural alteration of the nucleoplasm occurs. Cloning and sequencing indicate that the corresponding gene is NPL3/NOP3, which codes for a nucleolar/nuclear protein implicated in protein import into the nucleus (Bossie et al. (1992). Mol. Biol. Cell 3, 875-893) and in rRNA maturation (Russell and Tollervey (1992). J. Cell Biol. 119, 737-747). NPL3 includes bipartite RNA recognition motifs (RRM) and a Gly-Arg repeat domain, as in several nucleolar proteins. A point mutation adjacent to one of the RRM has been identified in the ts copy of the gene. Although this protein is not concentrated in nuclear pores, NPL3 is implicated in both import and export from the nucleus. Judging from the site of the npl3 mutation and since the block in RNA export can be detected prior to an obvious nuclear import defect in npl3, the defect in RNA export may be primary. Since other mutants that interrupt RNA export do not block protein import, the NPL3 protein itself appears to be implicated in protein import.