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Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins
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Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo
Gonzalo P Solis1, Arghavan Kazemzadeh2, Laurence Abrami3
1Translational Research Center in Oncohaematology, Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland. gonzalo.solis@unige.ch.
Nature Communications
|April 20, 2022
Summary
Peripheral membrane proteins (PMPs) are localized by palmitoyl acyltransferases (PATs). This study reveals PATs exhibit substrate selectivity, controlling PMP compartmentalization and offering new drug discovery avenues.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Peripheral membrane proteins (PMPs) are crucial for cellular functions and associate with membranes via post-translational modifications like S-palmitoylation.
- Palmitoyl acyltransferases (PATs) are enzymes responsible for S-palmitoylation, often localized to the Golgi apparatus for PMP modification and transport to destinations like the plasma membrane (PM).
- Limited information exists regarding the substrate specificity of different PATs, hindering a full understanding of PMP localization control.
Purpose of the Study:
- To investigate the inherent localization mechanisms of peripheral membrane proteins, specifically the Gαo subunit.
- To determine the substrate specificity of palmitoyl acyltransferases (PATs) in controlling protein compartmentalization.
- To establish a novel assay for probing PAT activity and substrate selectivity in intact cells.
Main Methods:
- Described the inherent partitioning of Gαo to the plasma membrane (PM) and Golgi, independent of Golgi-to-PM transport.
- Utilized a minimal N-terminal code within Gαo to govern its compartmentalization, demonstrating re-coding can alter localization.
- Established the S-palmitoylation at the outer nuclear membrane assay (SwissKASH) to probe PAT substrate specificity in live cells.
Main Results:
- Demonstrated that Gαo exhibits intrinsic localization to the PM and Golgi, governed by its N-terminus.
- Showcased that re-coding the Gαo N-terminus can shift its cellular localization.
- Revealed through the SwissKASH assay that compartment-specific PATs display significant substrate selectivity.
- Established that PAT substrate selectivity is a key mechanism for peripheral membrane protein (PMP) compartmentalization.
Conclusions:
- Peripheral membrane protein (PMP) localization is intrinsically governed by specific protein sequences and the substrate selectivity of compartment-localized palmitoyl acyltransferases (PATs).
- The developed SwissKASH assay provides a powerful tool for studying PAT activity and PMP localization in intact cells.
- These findings offer fundamental insights into protein trafficking and localization, paving the way for innovative drug discovery targeting membrane protein regulation.
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