Name: determining membrane protein topology using fluorescence protease protection (fpp)
Uploaded: 2015-04-20T14:00:00
Description: Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

We wish to thank the Calcium Imaging Core Facility at CMS for their help and guidance in image capture and analysis. This study was supported by the U.S. National Institutes of Health (NIH) HL102208 to N.A.B. This approach was originally pioneered by Holger Lorenz and Jennifer Lippincott-Schwartz at NIH.

1. Generation and Validation of Fluorescent Protein Chimeras
<ol>
	<li>Append green fluorescent protein (GFP) to the amino or carboxyl termini of the protein of interest using standard recombinant DNA strategies13. 
	NOTE: Although we have used GFP extensively, other variants such as cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), DsRed and mCherry can be employed. Improvements in fluorophore folding efficiency, brightness and photostability are constantly being made, and investigators...

<ol>
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The correct orientation and topology of membrane proteins is essential for their proper function. Despite the importance of understanding membrane protein topology, there are many proteins for which such data is completely lacking. FPP provides an easy and efficient way of determining membrane protein topology, and one that can be performed by most laboratories. The FPP approach affords significant advantages over previous methods for determining protein topology. For example, there is no need to have a panel of multiple...

The plasma membranes, as well as the numerous intracellular membranes, serve as barriers separating two aqueous compartments. In the case of the plasma membrane, the separation is between the outside and inside of the cell; for intracellular organelles it is between the cytoplasm and the organelle lumen. For example, the endoplasmic reticulum (ER) membrane separates an oxidizing environment within the lumen of the ER from a cytosolic reducing environment1. Membrane proteins are synthesized on ER-associated ribosomes, and achieve their final topology within the ER membrane2. The acquisition of appropriate membrane orientation and topology for proteins is critical for their normal function. Correct topology allows relevant domains of membrane proteins to interact with their binding partners, it allows critical post-translational modifications to occur, and in the case of plasma membrane proteins, allows the cell to interact with and respond to its environment. To fully appreciate the function of a membrane protein, it is clearly imperative to know how that protein is oriented with respect to the membrane within which it resides, i.e., its membrane topology. In addition to acquisition of basic scientific knowledge, understanding the topology of a membrane protein and which aspects of a protein surface are exposed to different environments has marked clinical implications since membrane proteins comprise the majority of pharmacological targets3. Until recently, approaches to determining membrane protein topology have required considerable investment in time and money or have required reagents that are difficult to come by.
Both experimental and in silico approaches have been employed to determine the membrane topology of proteins residing within the plasma membrane. Since the first predictions of membrane spanning domains based on the evaluated hydrophobicity of individual amino acids3, numerous predictive algorithms are now available on the internet, and simply require knowledge of the protein&#8217;s amino acid sequence. However, assumptions are often central to such modeling programs, assumptions that can lead to incorrect assignments of topology4,5. Moreover, while these computer-based predictions can tentatively assign membrane spanning regions, they do not always determine whether the amino or carboxy termini of proteins are in the cytoplasm, organelle lumen or cell exterior. Even with increased computational power, and the use of machine-learning algorithms6, such data is still a model, and must be validated using experimentally acquired data. Direct experimental determination of membrane topology has been undertaken using panels of monoclonal antibodies with known epitopes distributed throughout the protein, where assessment of their immunoreactivity has been made before and after cell permeabilization. This approach requires a set of antibodies, which may not be available for the protein of interest.
An alternative strategy is to engineer epitope tags such as myc or hemagglutinin (HA) into various locations throughout the protein, again followed by determination of immunoreactivity before and after membrane permeabilization. In addition to immunogenic tags, enzymatic tags (including alkaline phosphatase, &#946;-galactosidase, or &#946;-lactamase) and chemical modifications such as cysteine scanning have all been employed to determine membrane protein topology7,8. An additional method employed for topological mapping of plasma membrane proteins relies on a slightly different approach to epitope tags. In this method the tag sequence is the N-linked glycosylation consensus sequence NXS/T. Since glycosylation only occurs when such a sequence is present in the lumen of the biosynthetic pathway, the presence of the tag in a luminal versus a cytosolic compartment is easily observed as a mass shift on SDS-PAGE gels. Such an approach has been applied to the multispanning ion channel CFTR9. While all these approaches have been utilized, it is clear that they all require considerable investments in molecular biology to generate and sequence the manifold constructs.
To determine topological information regarding transmembrane proteins located within intracellular organelles has proven to be more challenging. The application of fluorescence based technologies however, has made the determination of membrane topology a lot simpler. The technique of bimolecular fluorescence complementation (BiFC) relies on the interaction between two non-fluorescent fragments of a fluorescent protein, restoring the fluorescent properties of that protein10. Although initially described for determining protein-protein interactions in vivo10, this approach has also been utilized to determine membrane protein topology in plant cells11. However this approach is also time intensive as it requires the generation not only of fusion proteins with the protein of interest but also a variety of fusion proteins targeted to the cytosol or organelle lumen, and requires knowledge of which intracellular membranes the protein of interest resides in.
An alternative simpler approach to determining membrane protein topology has been described12. The assay, Fluorescence Protease Protection (FPP) requires the generation of a fusion protein between GFP and the gene of interest. The approach is based on the relative accessibility of non-specific proteases to the GFP moiety; depending on whether GFP is protected from proteolysis by residing in the lumen of intracellular organelles or is exposed to proteolysis by being present in the cytosol. Thus, if the GFP moiety on the protein of interest faces the cytoplasm, it will be exposed to protease activity and the fluorescence signal lost. Conversely, if the GFP moiety on the protein of interest faces an environment &#39;protected&#39; from the protease (such as the Golgi lumen) then the fluorescence signal will persist.
To allow proteases to enter the cell, but not enter intracellular membrane compartments the cholesterol binding drug digitonin is used. Cholesterol is the dominant sterol in vertebrates, and is specifically enriched in the plasma membrane relative to intracellular compartments. The glycoside toxin, digitonin, is extracted from the plant Digitalis purpurea. Digitonin has an affinity for cholesterol rich membranes, where it leads to selective membrane permeabiliztion14,16 (Figure 1). In addition to allowing exit of small cytosolic components, digitonin permeabilization also permits the entry of exogenous molecules, such as proteinase K or trypsin. The FPP protocol takes advantage of the fact that the plasma membrane contains up to 80% of cellular cholesterol17, whereas other organelles, such as ER, Golgi, endosomes, mitochondria, which have very low cholesterol content, remain intact14. The selective incorporation of cholesterol into the plasma membrane has been observed in many eukaryotic cells, permitting the use of digitonin-dependent plasma membrane permeabilization in such diverse eukaryotic species as S. cerevisiae to human14,18. The FPP assay provides a simple, rapid and fairly robust means of determining (a) whether a protein is membrane bound/associated or freely diffusible in the cytosol and (b) which domain of a membrane protein faces the cytosol or organelle lumen. Should a membrane protein have multiple orientations, the signal will arise from the dominant form and minor forms will not be detected. While there can be some concern that the addition of a GFP moiety to the protein on interest may affect its function and/or subcellular localization, this is actually more theoretical than actual. Indeed many studies have clearly shown that the GFP tag does not alter the properties of the protein19,20.

<table border="0" cellpadding="0" cellspacing="0" width="370">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:111px;">Name</td>
			<td style="width:80px;">Company</td>
			<td style="width:116px;">Catalgue Number</td>
			<td style="width:64px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digitonin</td>
			<td>Calbiochem</td>
			<td align="right">300410</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cav1-GFP</td>
			<td>Addgene</td>
			<td align="right">44433</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pAcGFP-1 Golgi</td>
			<td>Clontech</td>
			<td align="right">632464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polylysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting Media</td>
			<td>Dako</td>
			<td>cS704</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining membrane protein topology using fluorescence protease protection (fpp)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Plasma Membrane Permeabilization
Efficient plasma membrane permeabilization is determined by the use of soluble fluorescent proteins (e.g., GFP, DsRed) (Step 4). These proteins, when expressed in cells, are free to diffuse in the cytosol, and are lost when the plasma membrane is permeabilized using digitonin (Figure 2). A complete disappearance of fluorescent signal should occur within 10-60 sec of digitonin application. Confirmation that the protein of inter...

Watch this Scientific Journal Video about Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP at JoVE.com

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

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