A vector for double epitope tagging with a recyclable marker.

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Multimeric protein complexes are crucial for cellular functions.
• Analyzing the composition and stoichiometry of these complexes is challenging.
• Understanding protein complex dynamics under varying conditions is vital.

Purpose of the Study:

• To design and validate a novel double epitope tagging vector.
• To facilitate the study of multimeric protein complex composition and stoichiometry.
• To enable the analysis of protein abundance within complexes.

Main Methods:

• Development of a vector for double epitope tagging at the protein's carboxy terminus.
• Incorporation of a universal HA1 epitope tag and a proteins-specific epitope tag.
• Utilized Cre-lox recombinase for marker recycling and tagged ribosome biogenesis proteins (Ytm1, Cic1, Brx1, Drs1).

Main Results:

• The double epitope tagging vector successfully tagged four proteins involved in ribosome biogenesis.
• A single anti-HA1 antibody detected all tagged proteins in crude lysates, revealing relative abundance.
• Drs1 was found to be reproducibly less abundant than other tagged proteins, suggesting regulatory implications.

Conclusions:

• The developed vector is effective for analyzing protein complex composition and stoichiometry.
• The findings on Drs1 abundance may offer insights into the control of ribosome biogenesis.
• This vector system holds potential for broader applications in studying diverse protein complexes.