Mapping a kingdom-specific functional domain of squalene synthase

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Squalene synthase is key in sterol biosynthesis, with distinct domains.
• Eukaryotic squalene synthases share architecture but differ in function.
• Previous studies suggested fungal C-terminal domains are vital for yeast complementation.

Purpose of the Study:

• Identify the specific motif in fungal squalene synthase responsible for complementation.
• Investigate the functional role of the enzyme's C-terminal domain.
• Determine the significance of the hinge region in sterol biosynthesis.

Main Methods:

• Utilized Saccharomyces cerevisiae with a squalene synthase knockout mutation.
• Expressed intact and chimeric squalene synthases from fungi, plants, and animals.
• Analyzed complementation phenotypes under varying gene expression levels.

Main Results:

• All tested enzymes partially complemented the mutation at low expression.
• High expression of non-fungal enzymes led to toxic intermediate accumulation.
• Complementation was restored by a 26-amino acid fungal hinge region linking catalytic and membrane domains.

Conclusions:

• The fungal hinge region is essential for squalene synthase function and complementation.
• This hinge domain plays a critical role in ergosterol multi-enzyme complex assembly in fungi.
• The hinge region's conservation suggests a fundamental functional importance across eukaryotes.