Nature Methods

A new method enabling the efficient isolation of protein complexes from mammalian cells is described in an article published online by Nature Methods this week. The technique can identify the partners in protein interaction networks, known as the 'interactome', which will help researchers to understand how specific proteins interact and function together in the cell.

A widely used affinity purification technique, tandem affinity purification, or TAP, uses specially designed purification tags as hooks to fish out a protein of interest and its interaction partners under gentle, close-to-physiological conditions that preserve binding interactions. The protein's interactome can then be identified with the aid of mass spectrometry. However, the method was originally developed in yeast, and the tag constructs were never optimized for their direct application in mammalian cells.

Giulio Superti-Furga and colleagues now describe a TAP-tag variant optimized for use in mammalian cells. Though they constructed and tested several variants, the purification tag with the most efficient properties consisted of two protein G immunoglobulin-binding units and a streptavidin-binding protein separated by a tobacco etch virus (TEV) protease cleavage site. This tag can be appended recombinantly to a protein of interest and used to isolate the protein and its interaction partners from cells in a two-step procedure.

Although the overall procedure is very similar to the yeast TAP method, the new tags enable the purification of mammalian protein complexes from a relatively small amount of cells and in quantities an order of magnitude higher than by using the original TAP tag. With this method, the authors conclude that "Large-scale approaches to explore the human proteome and cellular machinery should become more feasible."

CONTACT
Giulio Superti-Furga (Research Center for Molecular Medicine, Vienna, Austria)
Tel: +43 1 40160 70011; Email: gsuperti@cemm.oeaw.ac.at


News and Views: Pascal Braun (Harvard Medical School, Boston, Massachusetts, USA)
Tel: +1 617 582 8269; Email: pascal_braun@dfci.harvard.edu


Knocking out false positives in interaction proteomics

Nature Methods

A new method for identifying, with very high confidence, cellular proteins that naturally interact with each other is described in a paper published online this week in Nature Methods. By reducing the rate at which such protein interactions are falsely identified, the approach will help researchers construct more accurate 'wiring diagrams' that explain how proteins act together.

Matthias Mann and Matthias Selbach used antibody 'baits' immobilized on solid supports to fish out a target protein. Proteins that bind to the target protein under normal cellular conditions are thus also isolated along with the target protein itself. However, other proteins can cross-react with or nonspecifically bind to the antibody bait or solid support. These nonspecific interactions are also detected by the readout method and are known as false positives.

To control for this problem, the authors used a technique called RNA interference to knock out, or turn off, expression of the target protein in one population of cells. If the target protein is absent, only the nonspecific and cross-reactive binders will interact with the antibody bait. All of the isolated proteins from both the normal cell population and the knocked-out population are then identified by mass spectrometry. Proteins that are present in both cell populations are thus identified as false positives and discounted. This method is likely to become an important proteomic tool for studying human proteins, given the diverse range of antibodies and RNA interference reagents currently available.

CONTACT
Matthias Mann (Max Planck Institute of Biochemistry, Martinsreid, Germany)
Tel: +49 89 8578 2557; Email: mmann@biochem.mpg.de


News and Views: Pascal Braun (Harvard Medical School, Boston, Massachusetts, USA)
Tel: +1 617 582 8269; Email: pascal_braun@dfci.harvard.edu


Redefining receptor organization - again

Nature Methods

Serious doubt is cast on widely accepted models of how a crucial class of cell surface receptors organize themselves, in a report to be published in the December issue of Nature Methods.

Large proteins imbedded in the membranes of our cells mediate many of our most fundamental biological functions. Taste and smell result from small molecules in food and air binding to these proteins to trigger specific responses in the sensory cells that are then relayed to our brains. These proteins, called G-protein coupled receptors, also mediate many critical internal biological processes that involve communication between cells and are a principal target of many therapeutic drugs.

These receptors were thought to function like single sentinels manning the walls of an isolated outpost. Each looks for signals from the outside and relays them to innumerable others within the walls so that everyone can respond appropriately. In the 1990s evidence started to show that these receptors did not function as lone sentinels but as pairs who cooperated in relaying each outside signal. But it was difficult to determine if each sentinel was truly acting alone or in concert with another. Simon Davis and colleagues made adaptations to the principal experiment used to examine the organization of these cell membrane receptors in an attempt to make the results more conclusive. After testing the method on several different membrane receptors it appears that at least some of these receptor types only interact randomly and do not form true pairs.

In an accompanying News & Views Martin Lohse comments on these changing models of G-protein coupled receptor function and calls for a re-assessment of reports of dimer formation.

CONTACT
Simon Davis (University of Oxford, United Kingdom)
Tel: +44 1865 221 336; Email: simon.davis@ndm.ox.ac.uk


News and Views: Martin Lohse (University of Wuerzburg, Germany)
Tel: +49 931 201 48400; Email: lohse@toxi.uni-wuerzburg.de

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