Nucleocytoplasmic Distribution of Budding Yeast Protein Kinase A Regulatory Subunit Bcy1 Requires Zds1 and Is Regulated by Yak1-Dependent Phosphorylation of Its Targeting Domain

Autor: Maurizio David Baroni, Joakim Norbeck, Helmut Ruis, Paola Anghileri, Annalisa Ballarini, Paola Branduardi, Gerard Griffioen
Přispěvatelé: Griffioen, G, Branduardi, P, Ballarini, A, Anghileri, P, Norbeck, J, Baroni, M, Ruis, H
Rok vydání: 2001
Předmět:
Cytoplasm
Saccharomyces cerevisiae Proteins
Recombinant Fusion Proteins
Protein subunit
Molecular Sequence Data
Saccharomyces cerevisiae
Protein Serine-Threonine Kinases
Biology
Models
Biological
Fungal Proteins
Phosphoserine
Two-Hybrid System Techniques
Serine
medicine
Protein Isoforms
Amino Acid Sequence
Nucleocytoplasmic Communication
Phosphorylation
Protein kinase A
Molecular Biology
Adaptor Proteins
Signal Transducing
Cell Nucleus
Kinase
Intracellular Signaling Peptides and Proteins
Cell Biology
biology.organism_classification
Cyclic AMP-Dependent Protein Kinases
Protein Structure
Tertiary
Transport protein
Cell biology
Protein Subunits
Protein Transport
Cell nucleus
medicine.anatomical_structure
Microscopy
Fluorescence
Mutation
Mutagenesis
Site-Directed
budding yeast
protein kinase A
bcy1
subcellular distribution
zds1
Zdroj: Molecular and Cellular Biology. 21:511-523
ISSN: 1098-5549
DOI: 10.1128/mcb.21.2.511-523.2001
Popis: In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.
Databáze: OpenAIRE
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