RESUMEN
BACKGROUND: From 2003 through 2007, Vibrio cholerae serogroup O75 strains possessing the cholera toxin gene were isolated from 6 patients with severe diarrhea, including 3 in Georgia, 2 in Alabama, and 1 in South Carolina. These reports represent the first identification of V. cholerae O75 as a cause of illness in the United States. V. cholerae O75 was isolated from a water sample collected from a pond in Louisiana in 2004. Subsequently, 3 V. cholerae isolates from Louisiana (2 from patients with diarrhea in 2000 and 1 from a water sample collected in 1978) that had been previously reported as serogroup O141 were also discovered to be serogroup O75. RESULTS: All 8 patients who were infected with V. cholerae O75 were adults who became ill after consuming seafood; 2 had eaten raw oysters traced back to the Gulf Coast of the United States. All 10 isolates possessed the cholera toxin gene and were susceptible to 10 antimicrobials. One clinical isolate and 1 environmental (water) isolate had the same pulsed-field gel electrophoresis pattern; 4 clinical isolates shared a common pulsed-field gel electrophoresis pattern. CONCLUSIONS: The occurrence of these cases over many years and the concurrent identification of V. cholerae O75 in water from a Gulf Coast state suggest that these strains may survive for long periods in this environment. The patients' exposure histories suggest that infection can be acquired from consumption of raw oysters from the Gulf Coast. Clinicians and public health authorities should be vigilant for the occurrence of new toxigenic serogroups of V. cholerae that are capable of causing severe diarrhea.
Asunto(s)
Toxina del Cólera/biosíntesis , Cólera/epidemiología , Cólera/microbiología , Vibrio cholerae no O1/aislamiento & purificación , Vibrio cholerae no O1/metabolismo , Adulto , Anciano de 80 o más Años , Antibacterianos/farmacología , Toxina del Cólera/genética , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Femenino , Genotipo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Alimentos Marinos , Serotipificación , Sudeste de Estados Unidos/epidemiología , Vibrio cholerae no O1/clasificación , Vibrio cholerae no O1/efectos de los fármacos , Microbiología del AguaRESUMEN
Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin alpha, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin alpha, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.
Asunto(s)
Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Señales de Exportación Nuclear/fisiología , Unión Proteica/fisiología , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , alfa Carioferinas/químicaRESUMEN
Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha and beta. The importin alpha subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and auto-inhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin alpha export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha to the cytoplasm.
Asunto(s)
Saccharomyces cerevisiae/fisiología , alfa Carioferinas/fisiología , beta Carioferinas/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Núcleo Celular/fisiología , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Subunidades de Proteína/fisiología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , alfa Carioferinas/antagonistas & inhibidoresRESUMEN
Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.
Asunto(s)
Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae , alfa Carioferinas/metabolismo , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/genética , Mutación , Unión Proteica/genética , Temperatura , alfa Carioferinas/genéticaRESUMEN
Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.
Asunto(s)
Proteínas Fúngicas/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Carioferinas/química , Cinética , Proteínas Luminiscentes/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMEN
Nup98 is a component of the nuclear pore that plays its primary role in the export of RNAs. Nup98 is expressed in two forms, derived from alternate mRNA splicing. Both forms are processed into two peptides through autoproteolysis mediated by the C-terminal domain of hNup98. The three-dimensional structure of the C-terminal domain reveals a novel protein fold, and thus a new class of autocatalytic proteases. The structure further reveals that the suggested nucleoporin RNA binding motif is unlikely to bind to RNA. The C terminus also contains sequences that target hNup98 to the nuclear pore complex. Noncovalent interactions between the C-terminal domain and the cleaved peptide tail are visible and suggest a model for cleavage-dependent targeting of hNup98 to the nuclear pore.