Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when ...and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the C-terminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the C-terminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles.
The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving ...enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD. is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ. to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.
Freeze-fracture electron microscopy (FFEM) of kidney collecting duct, muscle, astrocytes in brain, and other mammalian tissues has revealed regular square arrays of intramembrane particles called ...orthogonal arrays of particles (OAPs). Their possible role in membrane structure and transport have been proposed, and their absence or decrease has been noted in a variety of hereditary and acquired diseases. A transgenic mouse lacking water channel AQP4 was used to show that AQP4 is the OAP protein. FFEM was done on kidney, skeletal muscle, and brain from AQP4 wild-type +/+, heterozygous +/- and knock-out -/- mice. The -/- mice did not express detectable AQP4 protein, but were grossly indistinguishable from +/+ mice. FFEM was done on blinded samples of kidney, brain and muscle from 9 mice. In all 6 kidney samples from +/+ and +/- mice, OAPs similar to those in AQP4-transfected CHO cells were found in basolateral membranes of collecting duct principal cells. In all muscle and brain samples from +/+ and +/- mice, OAPs of identical ultrastructure to those in kidney were seen, but in smaller patch sizes. OAPs were not seen in any sample from -/- mice. Label-fracture analysis using a peptide-derived AQP4 polyclonal antibody showed immunogold labeling of OAPs in AQP4-expressing CHO cells. These studies provide direct evidence that AQP4 is required for formation of OAPs and is a component of OAPs, thus establishing the identity and function of OAPs.
Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol ...facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems:Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.
To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the ...basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 ± 0.3× 10-4to 16.37 ± 0.5× 10-4cm/s at 20°C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 ± 0.3% under basal condition and decreased by 50% to 1.2 ± 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pfdecreased by ∼35% in 20-min histamine-stimulated cells. Both Pfand OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.
The yeastYPR192wgene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nineS. cerevisiaestrains. The osmotic water permeability coefficient (Pf) ofX. ...laevisoocytes expressing the gene cloned from the Σ1278b strain (AQY1-1) was 5.7 times higher than the Pfof oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1ΔCp), mediated an ∼3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pfmeasurements in yeast spheroplasts confirmed the presence of functional water channels in Σ1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.
The major health effect of uranium exposure has been reported to be chemical kidney toxicity, functional and histological damages being mainly observed in proximal tubule cells. Uranium enters the ...proximal tubule as uranyl-bicarbonate or uranyl-citrate complexes. The aim of our research is to investigate the mechanisms of uranium toxicity, intracellular accumulation and repartition after acute intoxication of rat renal proximal tubule epithelial cells, as a function of its chemical form.
Microscopic observations of renal epithelial cells after acute exposure to uranyl-bicarbonate showing the presence of intracellular precipitates as thin needles of uranyl-phosphate localized in cell lysosomes have been published. However the initial site of precipitates formation has not been identified yet: they could either be formed outside the cells before internalization, or directly inside the cells.
Uranium solubility as a function and initial concentration was specified by ICP-MS analysis of culture media. In parallel, uranium uptake and distribution in cell monolayers exposed to U-bicarbonate was investigated by nuclear microprobe analyses. Finally, the presence of uranium precipitates was tested out by scanning electron microscopic observations (SEM), while extracellular and/or intracellular precipitates were observed on thin sections of cells by transmission electron microscopy (TEM).
The Kidd (JK) blood group locus encodes a urea transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human ...erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 urea transporter, except for a Lys44→ Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/urea transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels inXenopus oocytes, the physiological Kidd/urea transporter HUT11A conferred a rapid transfer of urea (which was insensitive top-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the urea transport was poorly inhibited byp-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A transporter confers urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell urea transporter when expressed at unphysiological high levels.
Summary
Mps2 (monopolar spindle protein) is a coiled‐coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. ...We identified three proteins that interact with Mps2 in a two‐hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled‐coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2–Spc24 interaction by co‐immunoprecipitation in vivo
and by the in vitro interaction of recombinant proteins. Previous two‐hybrid
screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting
partners, and we verified these interactions in vivo by purification of TAP‐tagged
derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com‐ponents required for chromosome segregation. The Mps2–Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.
The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. ...laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.
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