Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis

There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent beta-aspartyl phosphate reaction intermediate. In this study, Asp-378 was replaced by Asn, Ser, and Glu, and the mutant ATPases were expressed in a temperature-sensitive secretion-deficient strain (sec6-4) that allowed their properties to be examined. Although all three mutant proteins were produced at nearly normal levels and remained stable for at least 2 h at 37 degrees C, they failed to travel to the vesicles that serve as immediate precursors of the plasma membrane; instead, they became arrested at an earlier step of the secretory pathway. A closer look at the mutant proteins revealed that they were firmly inserted into the bilayer and were not released by washing with high salt, urea, or sodium carbonate (pH 11), treatments commonly used to strip nonintegral proteins from membranes. However, all three mutant ATPases were extremely sensitive to digestion by trypsin, pointing to a marked abnormality in protein folding. Furthermore, in contrast to the wild-type enzyme, the mutant ATPases could not be protected against trypsinolysis by ligands such as MgATP, MgADP, or inorganic orthovanadate. Thus, Asp-378 functions in an unexpectedly complex way during the acquisition of a mature structure by the yeast PMA1 ATPase.     

Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase

The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.     

Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.     

Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.     

Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6-166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.     

The Saccharomyces cerevisiae early secretion mutant tip20 is synthetic lethal with mutants in yeast coatomer and the SNARE proteins Sec22p and Ufe1p

Tip20p is an 80 kDa cytoplasmic protein bound to the cytoplasmic surface of the endoplasmic reticulum (ER) by interaction with the type II integral membrane protein Sec20p. Both proteins are required for vesicular transport between the ER and Golgi complex. Recently, sec20-1 was found to be defective in retrograde transport. A collection of temperature-sensitive tip20 mutants are shown to be lethal in combination with ufe1-1, a target SNARE of the ER and ret2-1, yeast delta-COP. A subset of tip20 mutants was found to be lethal in combination with sec20-1, sec21-1, sec22-3 and sec27-1. Since all pairwise combinations of a tip20 mutant, sec20-1, and ufe1-1 are lethal, Tip20p and Sec20p might be part of the docking complex for Golgi-derived retrograde transport vesicles. Since carboxy-terminal tip20 truncations are lethal in combination with mutants in three coatomer subunits, Tip20p might be involved in binding or uncoating of COPI coated retrograde transport vesicles.     

PMR1, a Ca2+-ATPase in yeast Golgi, has properties distinct from sarco/endoplasmic reticulum and plasma membrane calcium pumps

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca2+-transporting ATPase in the Golgi, a hitherto unusual location for a Ca2+ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45Ca2+ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca2+ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 --> Glu and Asp-371 --> Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H+-ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca2+) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases. PMR1 therefore represents a new and distinct P-type Ca2+-ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca2+-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.     

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.     

Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3-1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61-2 allele. This is accompanied by the stabilization of the Sec61-2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61-2 strain at the permissive temperature of 25 degrees C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61-2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.     

Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.     

Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion

The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.     

Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome

A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.     

ATPase activity and molecular chaperone function of the stress70 proteins

The codons for the amino acid residues making up the proposed ATP-binding sites of the maize (Zea mays L.) endoplasmic reticulum and tomato (Lycopersicon esculentum) cytoplasmic Stress70 proteins were deleted from their respective cDNAs. The deletions had little effect on the predicted secondary structure characteristics of the encoded proteins. Both wild-type and mutant proteins were expressed in Escherichia coli and purified to electrophoretic homogeneity. The mutant recombinant proteins did not bind to immobilized ATP columns, had no detectable ATPase activity, and were unable to function in vitro as molecular chaperones. Additionally, the inability to bind ATP was associated with changes in the oligomerization state of the Stress70 proteins.     

Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.     

Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.     

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.     

Mutational analysis of the pea phytochrome A chromophore pocket: chromophore assembly with apophytochrome A and photoreversibility

Ten site-specific mutants of pea apophytochrome A were expressed in Saccharomyces cerevisiae and analyzed for chromophore assembly with apoprotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues located in the microenvironment of the chromophore attachment residue, which is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicating that there were no major structural perturbations in the apoproteins. However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three types: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted Pr and Pfr absorption maxima compared to the wild-type adduct, and mutants that are not photoreversible. From an analysis of the results, we concluded that the residues Asp-309, Arg-318, His-321, and Gln-326 are probably not catalytically involved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M. D., & Grimm, R. (1990) Z. Naturforsch, 45c, 987-998; Parker, W., et al. (1993) Bioconjugate Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critical for the spectral integrity and photoreversibility of phytochrome, but this residue is sterically important to the lyase activity. It appears that the role of the five amino acid residues in the N- and C-terminal vicinities of the chromophore binding Cys-323 is structural rather than catalytic for the ligation reaction.     

Characterization of yeast sar1 temperature-sensitive mutants, which are defective in protein transport from the endoplasmic reticulum

SAR1 encodes a low molecular weight GTPase that is essential in the early process of vesicular transport in the secretory pathway. By random and site-directed mutagenesis of the SAR1 gene, we have obtained three temperature-sensitive mutants, N132I, E112K, and D32G. They all show a defect in transport from the endoplasmic reticulum to the Golgi apparatus, and accumulate endoplasmic reticulum membranes at the restrictive temperature. This is consistent with our previous observations in vivo on a galactose-shutoff mutant as well as the in vitro results, and provides powerful tools for further genetic analyses.     

Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release

The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H+]-sensitive probes. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive fluorophore, was used for a two-stage delivery strategy. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Fluorescence ratio imaging and two independent calibration procedures were applied to determine the pH of the ER in situ. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. The ER was found to be highly permeable to H+ (equivalents), so that the prevailing [H+] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH.     

Cysteine-scanning mutagenesis around transmembrane segment III of Tn10-encoded metal-tetracycline/H+ antiporter

Each amino acid in the putative transmembrane helix III and its flanking regions (from Gly-62 to Tyr-98) of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) was individually replaced with Cys. Out of these 37 cysteine-scanning mutants, the mutants from G62C to R70C and from S92C to Y98C showed high or intermediate reactivity with [14C]N-ethylmaleimide (NEM) except for the M64C mutant. On the other hand, the mutants from R71C to S91C showed almost no reactivity with NEM except for the P72C mutant. These results confirm that the transmembrane helix III is composed of 21 residues from Arg-71 to Ser-91. The majority of Cys replacement mutants retained high or moderate tetracycline transport activity. Cys replacements for Gly-62, Asp-66, Ser-77, Gly-80, and Asp-84 resulted in almost inactive Tet(B) (less than 3% of the wild-type activity). The Arg-70 --> Cys mutant retained very low activity due to a mercaptide between Co2+ and a SH group (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Three of these six important residues (Ser-77, Gly-80, and Asp-84) are located in the transmembrane helix III and one (Arg-70) is located in the flanking region. These four functionally important residues are located on one side of the helical wheel. Only two of the residual 31 Cys mutants were inactivated by NEM (S65C and L97C). Ser-65 and Leu-97 are located on the cytoplasmic and periplasmic loops, respectively, in the topology of Tet(B). The degree of inactivation of these Cys mutants with SH reagents was dependent on the volume of substituents. In the presence of tetracycline, the reactivity of the S65C mutant with NEM was significantly increased, in contrast, the reactivity of L97C was greatly reduced, indicating that the cytoplasmic and periplasmic loop regions undergo substrate-induced conformational change in the mutually opposite direction.