The promyelocytic leukemia protein stimulates SUMO conjugation in yeast.

The promyelocytic leukemia gene was first identified through its fusion to the gene encoding the retinoic acid receptor alpha (RARalpha) in acute promyelocytic leukemia (APL) patients. The promyelocytic leukemia gene product (PML) becomes conjugated in vivo to the small ubiquitin-like protein SUMO-1, altering its behavior and capacity to recruit other proteins to PML nuclear bodies (PML-NBs). In the NB4 cell line, which was derived from an APL patient and expresses PML:RARalpha, we observed a retinoic acid-dependent change in the modification of specific proteins by SUMO-1. To dissect the interaction of PML with the SUMO-1 modification pathway, we used the budding yeast Saccharomyces cerevisiae as a model system through expression of PML and human SUMO-1 (hSUMO-1). We found that PML stimulated hSUMO-1 modification in yeast, in a manner that was dependent upon PML's RING-finger domain. PML:RARalpha also stimulated hSUMO-1 conjugation in yeast. Interestingly, however, PML and PML:RARalpha differentially complemented yeast Smt3p conjugation pathway mutants. These findings point toward a potential function of PML and PML:RARalpha as SUMO E3 enzymes or E3 regulators, and suggest that fusion of RARalpha to PML may affect this activity.

The interaction between Ran and NTF2 is required for cell cycle progression.

The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus. Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly. In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state. Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus. Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle. A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature. Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G(2) in a MAD2 checkpoint-dependent manner. These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo. Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest.

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.

Nuclear transport mechanisms.

The term nuclear transport, refers to the movement of a large variety of macromolecules both into and out of the nucleus. Transport must be extremely selective, yet also very efficient. A single type of channel, the nuclear pore complex, mediates all movement across the nuclear envelope. Selectivity is achieved through the use of families of soluble factors that target substrates for import and export and deliver them to their appropriate intracellular destinations. We now have a fairly detailed understanding of the basic mechanisms of protein import into the nucleus. Many of these same principles can be applied to protein export and perhaps RNA export. This review will summarize the current status of what is known about various transport pathways and highlight the questions that remain to be answered.

Genetic approaches to biochemical questions: insights into the functional requirements of proline 185 in the active site of human galactose-1-phosphate uridylyltransferase.

Saturating random mutagenesis at a given position within a polypeptide sequence can provide powerful insights into the functional requirements of the position. By coupling this genetic methodology with expression of human proteins in yeast, we and others have begun to ask pointed and important questions about the structure-function relationships of proteins associated with human genetic disease.

Performance of women candidates on the American Board of Internal Medicine Certifying Examination, 1973-1982.

Trends in the performances of female and male candidates taking the American Board of Internal Medicine Certifying Examination from 1973 through 1982 were examined. The mean scores of female candidates who graduated from medical schools in the United States or Canada and who were taking the examination for the first time improved from 428 to 470, and the percentage of those passing improved from 59% to 76%. The number of women taking the examination also increased markedly, by over 500%. Performance of female candidates remained slightly lower than that of male candidates, regardless of the quality of the residency training program or the medical school from which a candidate had graduated or the rating given a candidate by the director of the candidate's residency program. Except for the oldest candidates, age followed this pattern as well. Our findings suggest that the gender gap in scores on the Certifying Examination in Internal Medicine is narrowing.

Functional requirements of the active site position 185 in the human enzyme galactose-1-phosphate uridylyltransferase.

The active site of galactose-1-phosphate uridylyltransferase (GALT) includes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans. The crystal structure of the E. coli enzyme suggests that this proline is important in positioning the active site histidine (His-166) near the substrate. To examine the role of this proline in the homologous human sequence, we have performed saturating mutagenesis at Pro-185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system. Activity analyses in crude lysates indicated that only proline at position 185 produced wild-type levels of activity, although five other amino acids, Ala, Gly, Ser, Gln, and Glu, all produced partially active enzymes. Western blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity. All five active mutant proteins were purified and characterized with regard to specific activity, apparent Km for both substrates, and temperature-dependence of activity. Finally, modeling of these mutations onto the conserved E. coli active site structure was performed. Together, these results provide functional evidence demonstrating the critical role of Pro-185 in facilitating the transferase reaction.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

The mechanism of ran import into the nucleus by nuclear transport factor 2.

The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.

Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system.

Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT.

Functional analysis of the hydrophobic patch on nuclear transport factor 2 involved in interactions with the nuclear pore in vivo.

Nuclear transport factor 2 (NTF2) is a small homodimeric protein that interacts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interaction between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2-nucleoporin interaction. Analysis of these mutants in vivo demonstrates that the strength of this interaction can be significantly reduced without affecting cell viability. However, cells cease to be viable if the interaction between NTF2 and nucleoporins is abolished completely, indicating that this interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increased affinity for nucleoporins. Overexpression of the N77Y protein blocks nuclear protein import and concentrates Ran at the nuclear rim. These data support a mechanism in which NTF2 interacts transiently with FxFG nucleoporins to translocate through the pore and import RanGDP into the nucleus.

Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase.

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.

SAC3 may link nuclear protein export to cell cycle progression.

Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996) J. Cell Sci. 109, 1575-1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of SAC3 function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that SAC3 interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.