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Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane
Mansi R Khanna1, Bruce A Stanley, Graham H Thomas
1Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Background:
The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.
Results:
We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.
Conclusion:
A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.
Insights
Researchers developed a new method combining density gradient centrifugation and aqueous two-phase affinity purification (2PAP) for robust plasma membrane (PM) isolation in Drosophila. This technique enables improved proteomic analysis of PM proteins in this important model organism.
Area of Science:
- Cell Biology
- Proteomics
- Biochemistry
Background:
- The plasma membrane (PM) is crucial for cell-environment interactions but challenging to isolate due to low abundance.
- Existing Drosophila PM isolation methods rely on density gradient centrifugation, limiting genetic investigation.
- Aqueous two-phase affinity purification (2PAP) is effective for vertebrate PM isolation but not yet adapted for Drosophila.
Purpose of the Study:
- To adapt and optimize the 2PAP method for robust plasma membrane isolation in Drosophila.
- To facilitate proteomic studies of the Drosophila PM sub-proteome.
- To establish a reliable technique for isolating high-purity PM from Drosophila.
Main Methods:
- A novel combination of density gradient centrifugation and 2PAP was employed for PM isolation.
- The optimized protocol involved careful selection of lectins and partitioning conditions.
- Proteomic analysis using MudPIT was performed on isolated PM from Drosophila heads.
Main Results:
- The combined density gradient centrifugation and 2PAP method yielded a robust PM preparation.
- 432 proteins were identified, with 37% being integral membrane proteins.
- 22% of identified proteins were previously assigned to PM, and 34% are novel candidates.
Conclusions:
- The integrated density gradient centrifugation and 2PAP approach provides high-purity Drosophila PM, surpassing individual methods.
- This technique is foundational for proteomic investigations of PM in various Drosophila genetic backgrounds.
- Optimization of membrane partitioning and lectin selection are key for adapting this method to other systems.
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