Subunit compositions and catalytic properties of proteasomes from developmental temperature- sensitive mutants of Drosophila melanogaster.

Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.     

Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8 x 10(7) Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60 kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23 kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel.     

Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes

A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.     

Immunoaffinity isolation of Na+ channels from rat skeletal muscle. Analysis of subunits

Polyclonal antiserum and monoclonal antibodies raised against the sodium channel from rat skeletal muscle sarcolemma have been immobilized on Sepharose and used to immunoaffinity purify this channel directly from skeletal muscle without the intervening purification of surface membranes. These antibodies isolate a approximately 260-kDa protein from whole muscle, although each purifies predominantly a 150-kDa component when isolated sarcolemmal membranes are used as starting material. A 45-kDa band is also found in the material purified from sarcolemma but not that obtained from whole muscle. In addition, these immunoaffinity columns isolate a 38-kDa band from both whole muscle and sarcolemma that copurifies with the 260-kDa protein. In some preparations this component appears as two closely spaced bands of 37 and 39 kDa. These small subunits coelute with the 260-kDa subunit when thiocyanate gradients are used to displace protein bound to the immunoaffinity columns and behave as integral components of the sodium channel. Estimates of stoichiometry were made for the large and small subunits of the muscle channel protein. After correction for labeling efficiency, values consistent with a ratio of one 260-kDa subunit to one 38-kDa subunit were obtained. We conclude that the rat skeletal muscle sodium channel contains a large alpha subunit of approximately 260 kDa that is sensitive to proteolytic nicking during the isolation of sarcolemmal membranes. In addition, at least one 38-kDa beta subunit is associated with each alpha subunit in the native channel.     

A novel plant ferritin subunit from soybean that is related to a mechanism in iron release

Ferritin is a multimeric iron storage protein composed of 24 subunits. Ferritin purified from dried soybean seed resolves into two peptides of 26.5 and 28 kDa. To date, the 26.5-kDa subunit has been supposed to be generated from the 28-kDa subunit by cleavage of the N-terminal region. We performed amino acid sequence analysis of the 28-kDa subunit and found that it had a different sequence from the 26.5-kDa subunit, thus rendering it novel among known soybean ferritins. We cloned a cDNA encoding this novel subunit from 10-day-old seedlings, each of which contained developed bifoliates, an epicotyl and a terminal bud. The 26.5-kDa subunit was found to be identical to that identified previously lacking the C-terminal 16 residues that correspond to the E helix of mammalian ferritin. However, the corresponding region in the 28-kDa soybean ferritin subunit identified in this study was not susceptible to cleavage. We present evidence that the two different ferritin subunits in soybean dry seeds show differential sensitivity to protease digestions and that the novel, uncleaved 28-kDa ferritin subunit appears to stabilize the ferritin shell by co-existing with the cleaved 26.5-kDa subunit. These data demonstrate that soybean ferritin is composed of at least two different subunits, which have cooperative functional roles in soybean seeds.     

Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

Identification of the hyaluronan receptor for endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).     

The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle

Protein phosphatases-2A0, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2A0 and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2A0 and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2A0, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct. The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 +/- 6 kDa (2A0), 202 +/- 6 kDa (2A1) and 107 +/- 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2A0 and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits. Protein phosphatases-2A0, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 microM), histone H1 (A0.5 = 0.3 microM) and polylysine (A0.5 = 0.04 microM). Activation by protamine varied over 5-20-fold for phosphatase-2A0 and 5-7-fold for phosphatases-2A1 and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5-10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.     

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.     

Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex.

Western blot analysis, using a polyclonal antibody to the 240-kDa endogenous inhibitor of the 20 S proteasome, revealed that the inhibitor is a component of the 26 S complex. Although isolated inhibitor displayed a single 40-kDa band on SDS-PAGE, the antibody detected a 55-kDa component in the 26 S proteasome complex. Ubiquitin polyclonal antibody recognized the same 55-kDa component but did not react with free 40-kDa inhibitor subunit. Addition of purified 40-kDa inhibitor to a ubiquitin ligating system also generated the 55-kDa species. In crude erythrocyte extracts, most of the inhibitor migrated at 55 kDa in the presence of ATP but shifted to 40 kDa in the absence of ATP, consistent with removal of ubiquitin. It is suggested that ubiquitination of the inhibitor may be involved in regulating assembly and/or activity of the 26 S proteasome complex.     

Purification and characterization of Candida albicans 20S proteasome: identification of four proteasomal subunits

The 20S proteasome from yeast cells of Candida albicans was purified by successive chromatographic steps to apparent homogeneity, as judged by nondenaturing and denaturing polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 640 kDa by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave at least 10 bands in the range 20-32 kDa. Two-dimensional electrophoresis revealed the presence of at least 14 polypeptides. By electron microscopy after negative staining, the proteasome preparation appeared as typical symmetrical barrel-shaped particles. The enzyme cleaved the peptidyl-arylamide bonds in the model synthetic substrates Cbz-G-G-L-p-nitroanilide, Cbz-G-G-R-beta-naphthylamide, and Cbz-L-L-E-beta-naphthylamide (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities). The differential sensitivity of these activities to aldehyde peptides and sodium dodecyl sulfate supported the multicatalytic nature of this enzyme. Three proteasomal subunits were identified as alpha6/Pre5, alpha3/Y13, and alpha5/Pup2 by internal sequencing of tryptic fragments. Their sequences perfectly matched the corresponding deduced amino acid sequences of the C. albicans genes. A fourth subunit was identified as alpha7/Prs1 by immunorecognition with a monoclonal antibody specific for C8, the human proteasome subunit homologue. Treatment of the intact isolated 20S proteasome with acid phosphatase and Western blot analysis of the separated components indicated that the alpha7/Prs1 subunit is obtained as a multiply phosphorylated protein.     

Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE-cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N-ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme.     

A calcium-dependent protease associated with the neural cytoskeleton. Purification and partial characterisation

Calcium-dependent protease activity was found associated with a neurofilament-enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300-fold) by chromatography on organomercurial-Sepharose 4B, casein-Sepharose 4B, and Sepharose CL-6B. A cytosolic calcium-dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE-cellulose. Both cytoskeleton-bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+ and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1-0.2 mM Ca2+, the 68-kDa and 160-kDa components, and to a lesser extent the 200-kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton-bound protease needed two-fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton-bound protease in situ, i.e. before its extraction form the cytoskeleton by 0.6 M KCl, preferentially degraded the 160-kDa component in the presence of 0.1-0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160-kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.     

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein B_sarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein. Received: 18 May 1998 / Accepted: 5 August 1998     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.     

Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells

Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.