RESUMO
Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fuso Acromático/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia de Fluorescência , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
OBJECTIVES: To describe a method for measuring the direct and indirect costs to families of infants hospitalized with respiratory syncytial virus (RSV). METHODS: After pretesting and revising a questionnaire, a prospective survey was conducted in multiple tertiary-care hospitals with pediatric intensive care units. Eligible patients were infants less than 12 months old who had not received RSV prophylaxis and were hospitalized with a confirmed RSV infection. All English- and Spanish-speaking caregivers of eligible subjects were asked to participate in a face-to-face, structured interview on the day of hospital discharge regarding hospitalization-related direct and indirect costs. Thirty days later, caregivers were re-interviewed by telephone about their RSV-related costs during the elapsed month. The survey was initiated in February 2000 and continued through April 2001. RESULTS: In addition to the infants' parents, numerous adults visited 55% of hospitalized infants. In 17% of cases, nonparents missed work to visit the child. Volunteers watched siblings of 26% of the infants. Relying only on closed-ended questions about parents' costs during the hospitalization would have missed important information about child-care volunteers and types of expenses. Follow-up interviews revealed that RSV-related out-of-pocket expenses and missed work continued during the month following discharge. CONCLUSIONS: Survey instruments should be pretested with potentially eligible subjects. Open-ended questions are needed, because all costs cannot be anticipated. Respondents should be probed for details. This method revealed certain time and financial burdens during and after hospitalization that had not been previously reported in the literature.
