Functional and physical interactions between autonomously replicating sequence-binding factor 1 and the nuclear...

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Autonomously replicating sequence-binding factor 1 (Abf1p) regulates DNA replication, transcription, and silencing in Saccharomyces cerevisiae.
• Abf1p's carboxy-terminus contains two key regions, CS1 and CS2, crucial for its diverse nuclear functions.

Purpose of the Study:

• To investigate the role of the CS1 domain in the nuclear localization of Abf1p.
• To elucidate the mechanisms governing Abf1p's nuclear import and export.

Main Methods:

• Genetic analysis of Abf1p mutants.
• Cell biological studies including nuclear translocation assays.
• Protein localization studies using GFP fusion proteins.
• Biochemical assays to study protein-protein interactions.

Main Results:

• Mutations in the CS1 domain severely impair Abf1p's nuclear import, cell growth, and gene regulation.
• A heterologous nuclear localization sequence (NLS) rescues CS1 mutant defects.
• The CS1 domain mediates nuclear import dependent on Prp20p (Ran GEF).
• A specific CS1 mutation (K625I) leads to Crm1p-dependent nuclear export.
• Overexpression of Kap121p/Pse1p rescues the temperature-sensitive growth defect of the K625I mutant.
• Biochemical data suggest Abf1p possesses a second NLS interacting with Pse1p in a RanGTP-sensitive manner.

Conclusions:

• The CS1 domain is essential for the proper nuclear localization of Abf1p in yeast.
• Abf1p utilizes multiple, potentially overlapping, pathways for nuclear import, involving both CS1 and other regions.
• The interaction of Abf1p with the nuclear transport machinery is crucial for its diverse nuclear roles.