A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Ras proteins require posttranslational modifications for plasma membrane association.
• Key modifications include farnesylation, proteolysis, methylesterification, and palmitoylation.
• The CaaX box motif initiates these modifications via prenyl transferase recognition.

Purpose of the Study:

• To investigate the functional requirements for Ras membrane association.
• To assess the role of prenylation, palmitoylation, and basic amino acid stretches in yeast Ras2 protein function.
• To identify alternative carboxyl-terminal motifs that support Ras protein functionality.

Main Methods:

• Site-directed mutagenesis of the yeast Ras2 protein CaaX box.
• Analysis of Ras2 protein function in yeast strains with modified carboxyl-terminal sequences.
• Assessment of protein prenylation and palmitoylation status for mutant Ras2 proteins.

Main Results:

• Mutations replacing the CaaX box with a Lys-Leu-Ile-Lys-Arg-Lys sequence encode functional Ras proteins.
• One mutant, Ras2(CCIIKLIKRK), exhibited wild-type Ras2 function, while Ras2(SCIIKLIKRK) and Ras2(SSIIKLIKRK) showed reduced function at 30°C and thermosensitivity at 37°C.
• Mutants lacking basic residues (Ras2(CCIISIIS)) were non-functional, and extension mutants were not prenylated, but some were palmitoylated.

Conclusions:

• Functional Ras proteins in yeast can be generated by diverse carboxyl-terminal sequences beyond the canonical CaaX box.
• Posttranslational modifications, including palmitoylation, play crucial roles in Ras protein function, even with altered terminal motifs.
• This study highlights the adaptability of Ras protein targeting and function through varied modification pathways.