Abstract
BackgroundThe 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.ResultsWe 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.ConclusionA 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.
Highlights
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
Aqueous two-phase affinity partitioning I (6.3%PEG/ Dextran) Aqueous two-phase affinity partitioning (2PAP) separates membranes through their differing affinities for the polyethylene glycol and dextran phases coupled with the affinity of glycosylated surface proteins for lectins, and represents a quick and easy method to isolate plasma membrane (PM) proteins
We first attempted to isolate plasma membrane from Drosophila melanogaster using the Aqueous two-phase affinity purification (2PAP) method previously used to enrich for PM from rat brains [11]
Summary
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. To facilitate genetic investigation of activities contributing to the content of the PM subproteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. The plasma membrane (PM) and its associated proteins play an important role in determining how a cell interacts with its neighbours as well as how it responds to components of, and conditions in its extracellular environment. The amount of a protein at the cell surface is determined by its rate of delivery, internalization, recycling and degradation. All these parameters are subject to change during normal physio-.
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