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High-Throughput Isolation of Organelles

Thomas Squier, Principal Investigator

multiuse affinity probe (MAP), graphic
Depiction of multiuse affinity probe (MAP) bound to a tetracysteine tag sequence.

A long-term goal at Pacific Northwest National Laboratory (PNNL) is to develop affinity-based methods that permit the rapid, simultaneous isolation of cellular organelles in sufficient quantities for mass spectrometric analysis to identify protein compositional changes within individual organelles in response to environmental signals. Through the High-Throughput Isolation of Organelles project, PNNL researchers are working toward this goal by using the abundant protein, phospholamban (PLB), to isolate PLB-containing, endosomal-like vesicles and sarcoplasmic reticulum (SR), thereby testing the feasibility for using the proposed tags and isolation strategies for less abundant proteins.

schematic diagram
Top: Schematic diagram illustrating the capture of a tagged protein (red) and its associated binding partners onto FlAsH resin. Bottom: Eluted holoenzyme protein complex and sequence coverage of identified peptides using mass spectrometry. (A) Coomassie-stained SDS-PAGE gel of eluted protein complex associated with 4Cys-RNA polymerase (RpoA) bait protein (lane 2) relative to control nonspecific binding to beads (lane 1).

The biological problem driving this research is significant. Phospholamban, a small protein regulator of the SR in heart and skeletal muscle, has been extensively studied for its role in the progression of heart disease. Recently, we have discovered that phospholamban resides in small, endosomal-like vesicles of muscle cells during their early development stage, then moves into the SR membrane in differentiated muscle. This unexpected finding suggests that phospholamban may have a novel function in developing muscle that has not been appreciated before.

We are characterizing the protein composition of PLB-containing, endosomal-like vesicles in undifferentiated myocytes. Tagged phospholamban has been expressed in the C2C12 skeletal mouse muscle cell line, and during different stages of muscle development, we are isolating phospholamban-containing organelles for mass spectrometric analysis. Our isolation strategies involve comparing two different multi-affinity tags with cell-permeant ligands that allow proteins to be selectively probed in the living cell. Parallel capabilities are being developed for isolation of membrane protein complexes.

This work will provide new hypotheses regarding the role of PLB in developing heart and skeletal muscle. In addition, the occurrence of this protein in two kinds of organelles in different stages of cell development allows us an opportunity to compare the efficacy for isolation of the same protein from different organelles, small vesicles, and SR membranes.

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