Functional and physical interactions between autonomously replicating sequence-binding factor 1 and the nuclear transport machinery.
Autonomously replicating sequence-binding factor 1 (Abf1p) is a site-specific DNA binding protein in Saccharomyces cerevisiae that functions to regulate multiple nuclear events including DNA replication, transcriptional activation, and gene silencing. Previous work indicates that the multiple functions of Abf1p are conferred by the carboxy-terminus of the protein, which can be further dissected into two important clusters of amino acid residues (CS1 and CS2). Here we present genetic and cell biological evidence for a critical role of CS1 in proper nuclear localization of Abf1p. Mutations in CS1 cause severe defects in cell growth, nuclear translocation, and Abf1p-mediated gene regulation, which can be rescued by a heterologous nuclear localization sequence (NLS). In addition, the CS1-domain can mediate the import of a CS1-GFP fusion protein. Importantly, the CS1-mediated nuclear import depends on the Ran guanine nucleotide exchange factor Prp20p. Interestingly, a single amino acid change in CS1 (K625I) also causes the protein to be exported out of the nucleus via the Crm1p-dependent pathway. The temperature-sensitive growth phenotype of this particular mutant can be overcome by overexpression of Kap121p/Pse1p, a well-established nuclear transport receptor. Biochemical studies indicate that Pse1p binds to a region of Abf1p upstream of CS1 in a RanGTP-sensitive manner, suggesting that Abf1p has a second distinct NLS and can be imported into the nucleus by several overlapping pathways. We propose that the link between Abf1p and the nuclear transport machinery may also be important for partitioning multiple Abf1p-mediated nuclear processes.
Loch
Christian M
CM
Department of Biochemistry and Molecular Genetics, School of Medicine, PO Box 800733, University of Virginia, Charlottesville, VA 22908-0733, USA.
Mosammaparast
Nima
N
Miyake
Tsuyoshi
T
Pemberton
Lucy F
LF
Li
Rong
R
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D026901:Membrane Transport Proteins / Q000378:metabolism; D009154:Mutation; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D026901:Membrane Transport Proteins; C483822:PSE1 protein, S cerevisiae; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors