Measuring local protein concentration in living cells

The way proteins are organized inside a living cell remains largely unknown. Levy and colleagues present a new way to probe this organization and measure the extent to which cytoplasmic and membrane proteins encounter each other.

HFSP Long-Term Fellow Emmanuel Levy and colleagues
authored on Thu, 17 July 2014

Each cell in your body is an entire world: hundreds of millions of protein molecules are crowded into its microscopic confines. Some have permanent “jobs” in particular locations in the cell while others move around. Some form parts of long-lasting structures while others are produced for particular tasks and degraded soon after. To get an overall picture of this world, we need to know the rules that underlie its “social structure.” For example, do proteins that are located in the same compartment necessarily bump into each other more frequently than two proteins located in two different compartments?

Figure: Scientists classically measure cellular localization through the identification of a signal such as fluorescence, as illustrated on the left by an eye looking at the structure of a fluorescent protein. Here, we use a fluorescent protein in a completely different way that does not rely on its fluorescence. Instead, we ask “which other protein does the fluorescent protein encounter, and at which frequency, when expressed in yeast?” Thereby placing ourselves in the skin of the protein.

Levy and colleagues have been working on clarifying the answer to such questions. To do so, they are using a technique that involves splitting a protein that is vital for the cell’s survival into two halves and attaching each half to another protein. One of those proteins is the reporter, the other the target. If the target protein approaches the reporter, the two halves of the split protein will reunite, and the yeast strain will survive and grow. The more abundant the target protein is in the vicinity of the reporter, the healthier the growth of the yeast. Through this strategy, using cell growth as a readout, the authors measured the concentration of ~2,000 proteins in the yeast cytosol, with an accuracy that is comparable to that of mass spectrometry.

Subsequently, confining the reporter to cell membranes meant that protein concentrations were measured in the membrane environment. Expectedly, the reporter confined to membranes measured membrane proteins at a higher local concentration than the cytosolic reporter did. The difference in local concentration measured by both reporters could thus be used to locate proteins without a microscope.

More surprisingly, the local concentrations measured by the cytosolic and membrane reporters revealed that the chances of any two proteins meeting were first and foremost a product of their concentration in the cell. In other words, encounters between an abundant cytosolic protein and a low-abundance membrane protein are more frequent than encounters between two low-abundance membrane proteins. An analogy to this observation is that most people do not know all the neighbours living in their own building. Yet, most neighbours know the same famous (abundant) people such as the president, although the president resides in a different location.

That, along with their other findings, raises some very basic questions about how the cell truly functions. Randomness – a basic principle of evolution – appears to be built into our cells’ constitution. Nature is an opportunistic tinkerer that is just as likely to repurpose a tool that’s at hand as to evolve a new one. So rather than working on the assumption that the world of the cell is a highly organized realm in which every protein has a place in the overall structure, this research implies that scientists might begin to view the world of the cell as a fuzzy, “social network” type of organization in which chance meetings around the cell may determine how it functions.


High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation. Levy ED, Kowarzyk J, Michnick SW. Cell Rep. 2014 May 22;7(4):1333-40. doi: 10.1016/j.celrep.2014.04.009. Epub 2014 May 10.

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