Fluorescent sensors reporting the activity of ammonium transceptors in live cells

Abstract

Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (‘AmTrac’ and ‘MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track.DOI: http://dx.doi.org/10.7554/eLife.00800.001

Development of biosensors and their application in metabolic engineering

“…For instance, the vast number of metabolite-binding protein scaffolds found in nature has served as a resource, from which platform FRET sensors for metabolite accumulations can be designed. In addition to the numerous examples of ligands including sugar phosphates [17], amino acids [18,19], carboxylic acids [20], cofactors [21], ions [22][23][24], FRET sensors can also be adopted to sense intracellular redox status [25] and other events that may be otherwise difficult to monitor (e.g., macromolecular crowding) [26].…”

Nitrogen Nutrition, Its Regulation and Biotechnological Approaches to Improve Crop Productivity

“…Ammonia channel proteins have either 11 or 12 transmembrane helices and divided into structurally similar two halves with a Nout, C-in topology (long cytoplasmic C-terminal) but organized with pseudo-two-fold symmetry [186] [187]. Ammonium is transported along a pore in between these two halves of the protein [188] [189]. A cytosolic loop connects the two halves (5 th & 6 th helices) and the residues of the helices are involved in recruitment and transport of ammonium, but the mechanism involved is not known [184].…”

Getting to grips with ammonium

“…Moreover, these techniques have lacked the spatial and temporal resolution to study the workings of ammonium transport proteins in living cells. Now, in eLife , Wolf Frommer of the Carnegie Institution for Science and co-workers—including Roberto De Michele as first author—have developed a fluorescent biosensor to study ammonium transport proteins in vivo using fluorescence microscopy (De Michele et al, 2013). …”

“…High-resolution atomic structures have been obtained for two ammonium transport proteins—AmtB, which is found in bacteria (Khademi et al, 2004), and AMT-1, which is found in Archaea (Andrade et al, 2005)—and these structures indicate that the ammonium is transported along a pathway that lies between the two halves of the protein. Several residues situated in the fifth and sixth helices, and the highly mobile cytosolic loop that connects these two helices, are predicted to be involved in the recognition and transport of the ammonium (Andrade et al, 2005), but the details of the transport mechanism and its regulation are not fully understood.

Figure 1.Structures of transport and channel proteins.( A ) Representation of the ammonium transport protein studied by Frommer and co-workers (De Michele et al, 2013). This protein, which contains 11 transmembrane helices, transports ammonium into the cell (red arrow).

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“…( A ) Representation of the ammonium transport protein studied by Frommer and co-workers (De Michele et al, 2013). This protein, which contains 11 transmembrane helices, transports ammonium into the cell (red arrow).…”