Binding to cellular receptors results in increased iron release from transferrin at mildly acidic pH

In order to better understand the cellular delivery of iron from serum transferrin (Tf), we compared iron release from receptor-bound and free Tf. While free Tf did not release all iron until below pH 4.6, receptor-bound Tf released significantly more iron at mildly acidic pH, with essentially all iron released between pH 5.6 and 6.0. Since Tf is acidified to a minimum pH of 5.4 in K562 cells, this result accounts for the nearly complete extraction of iron from Tf by these cells. Comparison of fluorescence from Tf conjugated with lissamine rhodamine sulfonyl chloride (LRSC-Tf) free in solution and bound to receptor provides further evidence that the Tf receptor modulates low pH-mediated conformational changes in Tf. As pH was decreased from neutrality, the fluorescence of free LRSC-Tf began to increase below pH 6.2; the fluorescence of LRSC-Tf bound to human receptors did not increase until below pH 5.6. Binding to the Tf receptor, while facilitating iron release from Tf, appears to partially inhibit a conformational change that causes the increase in LRSC-Tf fluorescence at low pH. The fluorescence of human LRSC-Tf bound to murine receptors increases at a higher pH, 6.0, indicating that there are differences in conformational stabilization of Tf by receptors of different species. The results suggest that the Tf receptor, in addition to providing a means by which cells may internalize Tf, functions to increase the release of iron from Tf in the endosome.     

The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.     

A stability transition at mildly acidic pH in the alpha-hemolysin (alpha-toxin) from Staphylococcus aureus.

The effects of mildly acidic conditions on the free energy of unfolding (DeltaG(u)(buff)) of the pore-forming alpha-hemolysin (alphaHL) from Staphylococcus aureus were assessed between pH 5.0 and 7.5 by measuring intrinsic tryptophan fluorescence, circular dichroism and elution time in size exclusion chromatography during urea denaturation. Decreasing the pH from 7.0 to 5.0 reduced the calculated DeltaG(u)(buff) from 8.9 to 4.2 kcal mol(-1), which correlates with an increased rate of pore formation previously observed over the same pH range. It is proposed that the lowered surface pH of biological membranes reduces the stability of alphaHL thereby modulating the rate of pore formation.     

Hydrogen metabolism in a mildly acidic lake sediment (Knaack Lake)

Hydrogen metabolism was studied in anoxic Knaack Lake sediments by measuring the in situ concentrations of dissolved H₂, as well as the V_max, turnover rate constant, and K_m for H₂. The results show that the relatively low rate of H₂/CO₂-dependent methanogenesis is paralleled by a low turnover of the dissolved H₂ pool. H₂-dependent acetate formation did not appear to be of importance based on the discrepancy of the K_m for H₂ consumption between the sediment and the prevalent homoacetogenic microbial population. In this mildly acidic lake sediment, H₂ turnover apparently was limited by H₂ production from organic matter. During incubation of sediment under a gaseous headspace, H₂ escaped from the aqueous phase, and steady state concentrations of dissolved H₂ were significantly lower than under in situ conditions. H₂ concentrations increased upon addition of various organic substrates. H₂ turnover within the sediment appeared unrelated to the concentration of H₂ detected in the water column, especially in the epilimnetic water layers.     

Dynamic traffic through the recycling compartment couples the metal transporter Nramp2 (DMT1) with the transferrin receptor

Nramp2 (natural resistance-associated macrophage protein 2, also called DMT1 and Slc11a2) is a proton-dependent cation transporter, which plays a central role in iron homeostasis. To study the subcellular distribution and dynamics of the transporter, we generated a construct encoding the long splice variant of Nramp2 (isoform II) tagged with the hemagglutinin epitope on a predicted extracellular loop. Cells stably transfected with this construct revealed the presence of Nramp2 in both the plasma membrane and in an endomembrane compartment. By labeling the exofacial epitope with a pH-sensitive fluorescent indicator, we were able to establish that this variant of Nramp2 resides in a vesicular compartment with an acidic lumen (pH 6.2) and that acidification was maintained by vacuolar-type ATPases. Dual labeling experiments identified this compartment as sorting and recycling endosomes. Kinetic studies by surface labeling with 125I-labeled antibodies established that the fraction of endomembrane Nramp2 was approximately equal to that on the cell surface. The two components are in dynamic equilibrium: surface transporters are internalized continuously via a clathrin and dynamin-dependent process, whereas endosomal Nramp2 is recycled to the plasmalemma by a phosphatidylinositol 3-kinase-dependent exocytic process. Depletion of cholesterol had no discernible effect on Nramp2 internalization, suggesting that rafts or caveolae are not essential. Because the pH at the cell surface and in endosomes differs by >or=1 unit, the rates of transport of Nramp2 at the surface and in endomembrane compartments will differ drastically. Their subcellular colocalization and parallel trafficking suggest that Nramp2 and transferrin receptors are functionally coupled to effect pH-dependent iron uptake across the endosomal membrane.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.

We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell periphery. The ligand remained associated with such vesicles when the latter concentrated towards the cell center, before ending up in the juxtanuclear area. Throughout this vesicular trafficking pathway, clathrin colocalized with Tf. We conclude that Tf is processed intracellularly via small coated endosomal vesicles (CEV) and is not delivered into large tubular endosomes (CURL; compartment for uncoupling receptors and ligands), typical for ligand trafficking to lysosomes. By determining the kinetics of Tf internalization and by comparing the flow of Tf to that of a fluorescent glycolipid, it can also be concluded that CEVs display sorting and recycling properties, implying that small vesicles can be shed from or fuse with CEVs. Acidic pH does not prevent the formation of CEVs, but their intracellular movement, towards the cell center, is impeded.     

Rab22a regulates the sorting of transferrin to recycling endosomes

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16 degrees C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.     

The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected

We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min- 1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]- amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane.     

The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH

Pepsin is an aspartic protease that acts in food digestion in the mammal stomach. An optimal pH of around 2 allows pepsin to operate in its natural acidic environment, while at neutral pH the protein is denatured. Although the pH dependence of pepsin activity has been widely investigated since the 40s, a renewed interest in this protein has been fueled by its homology to the HIV and other aspartic proteases. Recently, an inactive pepsin conformation has been identified that accumulates at mildly acidic pH, whose structure and properties are largely unknown. In this paper, we analyse the conformation of pepsin at different pHs by a combination of spectroscopic techniques, and obtain a detailed characterisation of the intermediate. Our analysis indicates that it is the dominant conformation from pH 4 to 6.5. Interestingly, its near UV circular dichroism spectrum is identical to that of the native conformation that appears at lower pH values. In addition, we show that the intermediate binds the active site inhibitor pepstatin with a strength similar to that of the native conformation. Pepsin thus adopts, in the 6.5-4.0 pH interval, a native-like although catalytically inactive conformation. The possible role of this intermediate during pepsin transportation to the stomach lumen is discussed.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

A di-leucine sequence and a cluster of acidic amino acids are required for dynamic retention in the endosomal recycling compartment of fibroblasts

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56--84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M(15,16), DED(64--66), and LL(76,77). The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.     

Adaptations of sodium balance to low pH in a sunfish (Enneacanthus obesus) from naturally acidic waters

Summary Net sodium flux (J _net), sodium influx (J _in), and sodium efflux (J _out) were measured in two sunfish,Enneacanthus obesus (acid-tolerant) andLepomis gibbosus (less acid-tolerant), during 24 h exposure to soft water of pH's 4.0 and 3.5.E. obesus exhibited a mild transitory disturbance at both pH's caused by inhibitedJ _in and slightly stimulatedJ _out. Body and plasma ion concentrations ofE. obesus were measured weekly during exposures for 5 weeks to acidified artificial soft water (ASW). Body sodium concentration declined 30% during 2 weeks exposure to pH 3.5 but no further during the next three weeks. Exposure to pH 4.0 had no effect on body sodium concentration during the entire 5 weeks. Plasma sodium concentration declined 15% over a 3 week period at pH 3.5; there was no further change in the next two weeks. Plasma potassium concentrations, which were measured after 4 and 5 weeks at pH's 5.8 and 3.5 in ASW, were not significantly different. In a separate two week long experiment, plasma sodium concentration ofE. obesus in ASW was correlated with pH between pH's 3.5 and 7.5. This effect was mainly due to increases above pretreatment levels at pH 4.5 and above. Increased ambient sodium and calcium concentrations had no effect on body sodium concentration ofE. obesus at pH 5.8, but mitigated the effects of exposure to pH 3.5. Increased calcium concentrations up to 25 M at pH 3.5 increased body sodium concentration, but higher concentrations had no additional effect. Body potassium concentration and body water concentration ofE. obesus were linearly related to body sodium concentration under a wide variety of external conditions. This suggests the presence of a mechanism by whichE. obesus regulates plasma sodium levels and body fluid compartments in response to sodium loss. In contrast toE. obesus, L. gibbosus showed larger sodium losses at low pH resulting from greater acceleration ofJ _out; those exposed at pH 3.5 died in less than 12 h.L. gibbosus also had reduced body and plasma sodium concentrations at pH 4.5 and below; those at pH 4.0 were the lowest. Body potassium concentration ofL. gibbosus was reduced in those fish exposed to pH 4.0 and below, but body water was increased. Thus there are striking differences in the ability to regulate ion and water balance at low pH between an acid-tolerant specialist (E. obesus) and a less acid-tolerant generalist (L. gibbosus).Abbreviations ASW artificial soft water - WBM wet body mass - DBM dry body mass     

Microbial sulfate reduction in acidic (pH 3) strip-mine lakes

Abstract ³⁵SO₄ reduction was detected in slurries of sediments obtained from Reservoir 29 (pH 3.8) and Lake B (pH 6.2), two acid strip-mine lakes in Indiana. The rates varied seasonally and were higher in summer and fall than in the spring. The optimal pH for sulfate reduction in Reservoir 29 sediments was 5, but samples had increased activity at pH 7 within 24 h after adjusting the pH to this value. In Lake B, the optimal pH for sulfate reduction was the in situ pH (6.2). Sulfate reduction in both lakes was stimulated 2–3-fold by increasing p _H2. High concentrations (5 mM) of organic acids inhibited sulfate reduction at pH 3.8, but stimulation was observed at concentrations of 0.1 mM. Acid-volatile sulfides accounted for about 70% of the products of ³⁵SO₄ reduction.     

GGA proteins mediate the recycling pathway of memapsin 2 (BACE)

Memapsin 2 (BACE, beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta (A beta) and the progression of Alzheimer disease. Both memapsin 2 and APP are transported from the cell surface to endosomes where APP is cleaved by memapsin 2. We described previously that the cytosolic domain of memapsin 2 contains an acid cluster-dileucine motif (ACDL) that binds the VHS (Vps-27, Hrs, and STAM) domain of Golgi-localized gamma-ear-containing ARF-binding (GGA) proteins (He, X., Zhu, G., Koelsch, G., Rodgers, K. K., Zhang, X. C., and Tang, J. (2003) Biochemistry 42, 12174-12180). Here we report that GGA proteins colocalize in the trans-Golgi network and endosomes with memapsin 2 and a memapsin 2 chimera containing a cytosolic domain of a mannose-6-phosphate receptor. Depleting cellular GGA proteins with RNA interference or mutation of serine 498 to stop the phosphorylation of ACDL resulted in the accumulation of memapsin 2 in early endosomes. A similar change of memapsin 2 localization also was observed when a retromer subunit, VPS26, was depleted. These observations suggest that GGA proteins function with the phosphorylated ACDL in the memapsin 2-recycling pathway from endosomes to trans-Golgi on the way back to the cell surface.     

Identification of a pH 6.5 acetohydroxyacid synthetase inBacillus subtilis

Acetohydroxyacid synthetase activity of crude extracts ofBacillus subtilis grown in pH 7.0 minimal medium has a pH optimum of 7.5. However, the activity of extracts of cells grown in minimal medium of pH 6.0 shows a pH optimum of 6.5. Acetate or propionate induces formation of the pH 6.5 activity. Hydroxyapatite chromatography of a crude extract of cells grown in pH 7.0 medium shows one major and one minor peak of enzymatic activity. Both peaks have a pH optimum of 7.5–8.0. However, chromatography of an extract of cells grown in the presence of acetate reveals three peaks of activity: one major peak with a pH optimum of 6.5 and two minor peaks both having a pH optimum of 7.5–8.0.     

A helical hairpin region of soluble annexin B12 refolds and forms a continuous transmembrane helix at mildly acidic pH

Annexins are soluble proteins that are best known for their ability to undergo reversible Ca(2+)-dependent binding to the surface of phospholipid bilayers. Recent studies, however, have shown that annexins also reversibly bind to membranes in a Ca(2+)-independent manner at mildly acidic pH. We investigated the structural changes that occur upon pH-dependent membrane binding by performing a nitroxide scan on the helical hairpin encompassing helices A and B in the fourth repeat of annexin B12. Residues 251-273 of annexin B12 were replaced, one at a time, with cysteine and then labeled with a nitroxide spin label. Electron paramagnetic resonance (EPR) mobility and accessibility analyses of soluble annexin B12 derivatives were in excellent agreement with the known crystal structure of annexin B12. However, EPR studies of annexin B12 derivatives bound to membranes at pH 4.0 indicated major structural changes in the scanned region. The helix-loop-helix structure present in the soluble protein was converted into a continuous transmembrane alpha-helix that was exposed to the hydrophobic core of the bilayer on one side and exposed to an aqueous pore on the other side. Asp-264 was on the hydrophobic membrane-exposed face of the amphipathic transmembrane helix, thereby suggesting that protonation of its carboxylate group stabilized the transmembrane form. Inspection of the amino acid sequence of annexin B12 revealed several other helical hairpin regions that might refold and form continuous amphipathic transmembrane helices in response to protonation of Asp or Glu switch residues on or near the hydrophobic face of the helix.     

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca²⁺ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca²⁺ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.