Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The overall goal of this procedure is to determine the topology of integral membrane proteins. This is achieved by expression of effusion, protein between green fluorescent protein or GFP and the amino or carboxyl tails of the membrane. Protein of interest in cells for control purposes, soluble fluorescent proteins such as RFP can be expressed.

Next, the plasma membrane is subject to selective permeation. Using the detergent dig, soluble fluorescent proteins will exit the cell, whereas membrane-bound fluorescent proteins will be retained within the cell. The final step is application of a protease to the perme cells and determination of retention or loss of fluorescent signal.

Ultimately, changes in fluorescence intensity observed using fluorescence microscopy imaging is used to show the topology and orientation of integral membrane proteins. The main advantage of this technique over existing methods, such as epitope mapping, is that the procedure doesn't require a lot of specialized equipment or various reagents that may be hard to come by. This method can help answer key questions in membrane protein biology, such as which domains of an integral membrane protein may be located within the cytosol versus which domains may be located within the lumen of organelles such as the endoplasmic reticulum gold G or endosomes.

Begin this procedure with the generation and validation of fluorescent protein KY as described in the text protocol. Then transfect the fluorescent protein KY into human embryonic kidney cells or HEK 2 93 cells using any standard high efficiency, low toxicity transfection method, including lipid, viral, or electroporation based technologies to generate transient or stable expression, cells are then plated on polylysine coated cover slips. Maintain the cells for five to 48 hours in appropriate tissue culture media in 37 degrees Celsius in a carbon dioxide capable tissue culture incubator.

Monitor the cells for expression of the GFP fusion protein using epi fluorescence microscopy, and the intrinsic fluorescent signal of GFP fluorescent signal usually reaches a maximum within 24 to 72 hours. Following transfection. Use the cells when the fluorescent signal reaches a maximum level protein expression can also be monitored by immuno blot analysis using antibodies directed against either the protein of interest or the GFP moiety.

To begin set up the gravity fed perfusion system for treating the cells. Prepare 50 milliliter syringes for solutions with polyethylene tubing that is connected to independent reservoirs and to a manifold with a single outlet at the bath. Position a suction pipette to maintain a constant bath volume and achieve solution exchange by manually switching between perfusion reservoirs for plasma membrane permeation.

First, remove the cell culture medium from the cells. Wash the cells three times for one minute each. In KHM buffer, place the cover slips on a fluorescence microscope stage and collect the first image, which represents the control nonpermeable image to selectively perme the plasma membrane.

Add digit toin in KHM buffer to the cells, perfuse the cells with buffer plus digit toin at a rate of five milliliters per minute for 10 to 60 seconds. Confirmation that the plasma membrane is adequately perme is easily performed by expression of a soluble GFP or RFP molecule, which will readily diffuse out of the cell following membrane. Permeation then confirm that the GFP protein chimer of interest is membrane integrated by confirming cellular retention of the fluorescent signal following digon and permeation of the plasma membrane.

Next, express an organal specific fluorescent protein such as a mitochondrial protein as referenced in the text protocol. Determine optimal expression conditions as before. Confirm that the dig and concentration is appropriate for the cell type employed by ensuring that the morphology of intracellular organ canals remains constant over prolonged exposure.

To dig toin. After washing the cells in KHM buffer, add protease directly onto the cells. Peruse the cells at a flow rate of five milliliters per minute, using a gravity fed perfusion system to continuously bathe the preparation.

Next, determine whether the fluorescent signal persists or degrades. Collect fluorescent microscopy images under the appropriate filter sets by using the video image capture function on a computer controlled fluorescent microscope. Quantify fluorescent signal intensities as a function of incubation condition.

Obtain the mean pixel intensity within a region of interest that encloses an individual cell. A loss of greater than 90%of the initial signal intensity over 30 to 60 seconds is consistent with proteolytic degradation of the GFP protein ky. A complete disappearance of soluble fluorescent signal should occur within 10 to 60 seconds of digit toin application.

Confirmation that the protein of interest is associated with intracellular organelles is achieved by noting the retention of fluorescent signal following digon and permeation. The carboxyl tail of membrane anchored kinase LTK two was fused to GFP and expressed transiently fluorescent ltk. Two GFP signal was lost rapidly following addition of proteinase K indicating the location of the carboxyl terminus of ltk two is within the cytosol.

Cavi and GFP is a plasma membrane protein with a GFP moiety attached to the cytosolic domain. As with LTK two, the signal from cavi and GFP is digit toin insensitive, but sensitive to the subsequent addition of protease DS red mito encodes red fluorescent protein fused to the mitochondrial targeting sequence from cytochrome C oxidase following digon permeation, as well as addition of proteinase K.The fluorescence associated with DS red mito remains stable. This is consistent with the Fluor four being in the mitochondria where it is not exposed to the cytosol.

When the fluorescent moiety attached to a membrane protein is facing the cell exterior, as in GPI anchored prion protein attached to yellow fluorescent protein. Proteinase treatment should rapidly quench the fluorescent signal without the prior need for dig imp permeation as expected. Following proteinase treatment, the extracellular fluorescent signal disappeared rapidly for cells expressing this construct.

Once mastered, this technique can be performed in one to two hours if done properly. After watching this video, you should have a greater understanding of how to determine the topology of your membrane protein of interest.