Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO 2 -triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO 2 -related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO 2 -triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO 2 -related transport processes, a structure built up by the tetramer is the basis of this function.Water moves across biological membranes by diffusion. In most living organisms, the rate of water diffusion can be increased via pore-forming transmembrane proteins, the socalled aquaporins. These consist of six-membrane-spanning helices; N and C termini of the proteins face the cytosol. The helix-connecting loops B and E are themselves short helices that dip into the membrane from opposite sides and form the water-conducting channel (1, 2). Aquaporin monomers can assemble into tetramers (3, 4). Evidence for their function as water transport facilitators was observed by results from experiments using heterologous expression systems, such as Xenopus laevis oocytes (5). Despite this initially detected function, a facilitated membrane transport for glycerol or volatile substances like CO 2 or NH 3 was postulated (6 -9). Plant aquaporins were subdivided into protein groups according to the cellular location in which they have been initially detected and on the basis of sequence similarities (10). Accordingly, the PIPs (plasma membrane-intrinsic proteins) were split into two major groups, the PIP1 and PIP2 aquaporins. Compared with PIP1 proteins, PIP2 proteins have a shorter N-terminal extension and a longer C terminus (11)(12)(13)(14). In general, plant PIP2 proteins have been shown to facilitate membrane water transport in heterologous expression systems; however, PIP1 proteins display low or no activity in this respect (8,11,(15)(16)(17)(18). For the human AQP1 as well as for the tobacco PIP1 aquaporin NtAQP1, an increased cellular acidification rate under CO 2 -enriched buffer was obtained in oocytes expressing the respective aquaporin in addition to a carbonic anhydrase (9,19,20). The observations on NtAQP1, together with the fact that plants deficient in NtAQP1 express...

Signal transduction in prokaryotes is frequently accomplished by two-component regulatory systems in which a histidine protein kinase is the sensory component. Many of these sensory kinases control metabolic processes that do not show an obvious requirement for inhomogeneous distribution within bacterial cells. Here, the sensory kinases DcuS and CitA, two histidine kinases of Escherichia coli, were investigated. Both are membrane-integral and involved in the regulation of carboxylate metabolism. The two-component sensors were fused with yellow fluorescent protein (YFP) and live images of immobilized cells were obtained by confocal laser fluorescence microscopy. The fluorescence of the fusion proteins was concentrated at the poles of the cells, indicating polar accumulation of the sensory kinases. For quantitative evaluation, line profiles of the imaged fluorescence intensities were generated; these revealed that the fluorescence intensity of the polar bright spots was 2.3-8.5 times higher than that of the cytoplasm. With respect to the cylindrical part of the membrane, the values were lower by about 40 %. The polar accumulation was comparable to that of methyl-accepting chemotaxis proteins (MCPs) and MCP-related proteins. The degree of DcuS-YFP localization was independent of the presence of MCP and the expression level of dcuS-yfp (or DcuS concentration). The presence of effector (fumarate or citrate, respectively) increased the polar accumulation by more than 20 %. Cell fractionation demonstrated that polar accumulation was not related to inclusion body formation. Therefore, sensory kinases DcuS and CitA, which regulate metabolic processes without obvious polar function, exhibit polar accumulation.

This study introduces a method to detect individual oxygen molecules by fluorescence microscopy of single hemocyanins. These respiratory proteins from a tarantula bind oxygen with high affinity. A spectrometric signature of the oxygenated protein is transferred to an attached fluorescence label, which can be detected at the single-molecule level. This technique opens new perspectives for the development of small and sensitive oxygen sensors as well as for the investigation of cooperative oxygen binding in respiratory proteins.