Protein Of Apparent Molecular Mass Research Articles

AP-3: an adaptor-like protein complex with ubiquitous expression.

We have identified two closely related human proteins (sigma3A and sigma3B) that are homologous to the small chains, sigma1 and sigma2, of clathrin-associated adaptor complexes. Northern and Western blot analyses demonstrate that the products of both the sigma3A and sigma3B genes are expressed in a wide variety of tissues and cell lines. sigma3A and sigma3B are components of a large complex, named AP-3, that also contains proteins of apparent molecular masses of 47, 140 and 160 kDa. In non-neuronal cells, the 47 kDa protein most likely corresponds to the medium chain homolog p47A, and the 140 kDa protein is a homolog of the neuron-specific protein beta-NAP. Like other members of the medium-chain family, the p47A chain is capable of interacting with the tyrosine-based sorting signal YQRL from TGN38. Immunofluorescence microscopy analyses show that the sigma3-containing complex is present both in the area of the TGN and in peripheral structures, some of which contain the transferrin receptor. These results suggest that the sigma3 chains are components of a novel, ubiquitous adaptor-like complex involved in the recognition of tyrosine-based sorting signals.

Characterization of Densin-180, a New Brain-Specific Synaptic Protein of theO-Sialoglycoprotein Family

We purified an abundant protein of apparent molecular mass 180 kDa from the postsynaptic density fraction of rat forebrain and obtained amino acid sequences of three tryptic peptides generated from the protein. The sequences were used to design a strategy for cloning the cDNA encoding the protein by polymerase chain reaction. The open reading frame of the cDNA encodes a novel protein of predicted molecular mass 167 kDa. We have named the protein densin-180. Antibodies raised against the predicted amino and carboxyl sequences of densin-180 recognize a 180 kDa band on immunoblots that is enriched in the postsynaptic density fraction. Immunocytochemical localization of densin-180 in dissociated hippocampal neuronal cultures shows that the protein is highly concentrated at synapses along dendrites. The message encoding densin-180 is brain specific and is more abundant in forebrain than in cerebellum. The sequence of densin-180 contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. This arrangement of domains is similar to that of several adhesion molecules, in particular GPIbalpha, which mediates binding of platelets to von Willebrand factor. We propose that densin-180 participates in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses.

Recruitment of Activated p56lck on Endosomes of CD2-triggered T Cells, Colocalization with ZAP-70

We have previously established that upon CD2 activation of T cells, p56(lck) showed a transient increase in its kinase activity and was partially internalized. Here we studied the possibility that p56(lck) could retain its kinase activity in the endosomes of CD2-triggered cells. T cells were fractionated on a sucrose gradient, and the endosomal fraction was isolated. In CD2-triggered cells, part of Lck was internalized and presented a maximal kinase activity in the endosome-enriched fraction after 5 min, decreasing thereafter. In the endosomal fraction of activated cells, four tyrosine-phosphorylated proteins of apparent molecular masses of 30, 40, 56, and 70 kDa were detected. We demonstrated that the protein tyrosine kinase ZAP-70 was recruited to the endosomal fraction upon CD2 stimulation with kinetics similar to that of p56(lck), suggesting that recruitment of protein tyrosine kinases to endosomal vesicles could promote specific transduction signals at the intracellular level.

The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

The Regions of the Fe65 Protein Homologous to the Phosphotyrosine Interaction/Phosphotyrosine Binding Domain of Shc Bind the Intracellular Domain of the Alzheimer's Amyloid Precursor Protein

Fe65 is a protein mainly expressed in several districts of the mammalian nervous system. The search of protein sequence data banks revealed that Fe65 contains two phosphotyrosine interaction (PID) or phosphotyrosine binding (PTB) domains, previously identified in the Shc adaptor molecule. The two putative PID/PTB domains of Fe65 were used to construct glutathione S-transferase-Fe65 fusion proteins. Co-precipitation experiments demonstrated that the Fe65 PID/PTB domains interacted with several proteins of apparent molecular mass 135, 115, 105, and 51 kDa. The region of Fe65 containing the PID/PTB domains was used as a bait to screen a human brain cDNA library in yeast by the two-hybrid system. Three different cDNA clones were isolated, two of which contain overlapping segments of the cDNA encoding the COOH terminus of the Alzheimer's beta-amyloid-precursor protein (APP), that represents the short intracellular domain of this membrane protein. The third clone contains a cDNA fragment coding for the COOH terminus of the human counterpart of a mouse beta-amyloid-like precursor protein. The alignment of the three APP encoding cDNA fragments found in the screening suggests that the region of APP involved in the binding is centered on the NPTY sequence, which is analogous to that present in the intracellular domains of the growth factor receptors interacting with the PID/PTB domain of Shc.

Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate.

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.

Stimulation of a Ca2+-Calmodulin-activated Histone 3 Arginine Kinase in Quiescent Rat Heart Endothelial Cells Compared to Actively Dividing Cells

A Ca(2+)-calmodulin-activated histone 3 kinase was partially purified from nuclear extracts of dividing and quiescent rat heart endothelial cells. The histone 3 phosphorylating activity was 20-100-fold higher in quiescent than in dividing cells. Base hydrolysis followed by amino acid analysis revealed that histone 3 was phosphorylated on arginine. Further investigations were conducted to determine whether phosphorylation of histone 3 also occurred in vivo. Cells were incubated for 3 h in a phosphate-free medium supplemented with [32P]phosphoric acid. It was observed that the nuclear content of arginine-phosphorylated histone 3 was considerably higher in quiescent than in dividing rat heart endothelial cells. The histone 3 arginine kinase is a component of a complex containing a Ca(2+)-dependent calmodulin-binding protein of apparent molecular mass of 85 kDa. Using polyclonal antibodies to an 85-kDa protein, also the major Ca(2+)-dependent calmodulin-binding component of the histone 3 arginine kinase from calf thymus, an immunoreactive protein of identical apparent molecular mass was found to be present in equal amounts both in dividing and quiescent cells. We propose that the 85-kDa protein is either the histone 3 arginine kinase or one of its subunits and that phosphorylation of histone 3 is involved with cell cycle exit in eukaryotes.

Mechanism of Activation and Functional Significance of the Lipolysis-Stimulated Receptor. Evidence for a Role as Chylomicron Remnant Receptor

In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

Inhibitory Effect on the Lipolysis-stimulated Receptor of the 39-kDa Receptor-associated Protein

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

Effects of growth hormone antagonists on 3T3-F442A preadipocyte differentiation

We have previously shown that a bovine (b) GH antagonist, bGH-M8, which possesses three amino acid substitutions in its third alpha-helix, inhibits mouse 3T3-F442A preadipocyte differentiation. In the current studies, we used the bGH and human (h) GH analogs with single amino acid substitution, bGH-G119R and hGH-G120R, for determining their biological activity using the preadipocyte differentiation assay. Short-term and long-term GH-inducible events were studied during adipose differentiation, including late marker gene expression (adipocyte protein 2), immediate early gene induction (c-fos), and tyrosine phosphorylation of intracellular proteins. The results demonstrated that these GH analogs not only failed to induce these three events, but also antagonized GH induction of c-fos expression and phosphorylation of proteins of apparent molecular mass of 95 kDa. Our present study agrees with the notion that GH must bind to the GH receptor via site one and with a second GH receptor molecule (or with some yet unidentified 'second target') through GH binding site two. This interaction is important for subsequent GH-dependent biological events.

Salt‐dependent Protein Composition in the Halotolerant Green Alga, Dunaliella parva

AbstractThe extremely halotolerant green alga, Dunaliella parva, tolerates salt concentrations from 0.3 to 3.0 M NaCl. Effects of long‐term adaptation to five distinct salinities were analyzed. Salt‐dependent differences of physiological parameters such as growth rate, pigments, quantitative protein contents, and gas exchange were measured; furthermore the qualitative protein composition in salt‐adapted cells was investigated using SDS‐polyacrylamide gel electrophoresis. Proteins of apparent molecular masses of 26, 35, 39, 50, and 63 kDa were induced or intensified with an increase in external sodium chloride concentration whereas proteins of 85 and 101 kDa were diminished in high salt algae. After selective staining, four modifications of glycoproteins were observed. A glycoprotein of 96 kDa was produced exclusively in low salt cells whereas glycosylations of 105, 135, and 260 kDa were induced by high salt concentrations.

The gene for Campylobacter trigger factor: evidence for multiple transcription start sites and protein products

A gene encoding a protein of apparent molecular mass 56 kDa that shares 31% identity with the amino acid sequence of trigger factor from Escherichia coli (a protein thought to be involved in cell division), was cloned from Campylobacter jejuni NCTC 11168. The clone was selected from a lambda ZAP II genomic DNA library following an immuno-screen using antiserum raised against glycine-extractable proteins from C. jejuni. The gene has two potential initiation codons, giving rise to two possible nested protein products. Complex differential growth-phase-dependent transcripts give rise to these products.

Characterization of Lipid Exchange Proteins Isolated from Small Intestinal Brush Border Membrane

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.

Photoaffinity labelling of the ATP-binding sites of two Ca 2+,Mg-ATPase isoforms in pancreatic endoplasmic reticulum

Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca 2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca 2+, Mg-ATPase activity only after UV irradiation and the inhibition was prevented by inclusion of 5 mM ATP in the labelling reaction. Rough ER proteins of apparent molecular masses 141, 111, 100, 84, 69, 55 and 47 kDa were detected following photoaffinity-labelling with 8-azido-[α- 32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca 2+,Mg-ATPase isoforms previously demonstrated immunologically [1]. Immunoprecipitation of rough ER proteins by a SERCA-2b-specific antibody showed that the two ATPase bands were photoaffinity-labelled. Photoaffinity labelling of the 111 and 100 kDa proteins was: (a) abolished when Ca 2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca 2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca 2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.

A group of integral membrane proteins of the rat liver Golgi contains a conserved protein of 100 kDa.

Rat liver Golgi membranes were washed with KCl and urea, and a polyclonal antiserum that stained the Golgi complex by immunofluorescence microscopy was raised. A group of proteins of apparent molecular mass 500 kDa, 200 kDa and 100 kDa were identified by immunoblotting with the antiserum, and were enriched in the Golgi membrane fraction. These proteins were also localised to the Golgi by immunofluorescence microscopy with affinity-purified antibodies. They are integral membrane proteins, and protease digestion experiments suggested that they are not exposed on the cytoplasmic face of the Golgi membrane. Immunofluorescence microscopy showed that staining of the Golgi complex by antibodies to the 100 kDa Golgi protein can be demonstrated among a wide range of mammalian species. This conservation may point to an important structural or functional role for the molecule. When the 100 kDa protein was reduced with dithiothreitol it was no longer recognised by the anti-Golgi antiserum. During phase separation in Triton X-114 the 100 kDa protein partitioned into the aqueous phase, rather than into the detergent phase, suggesting that it has a large luminal domain of hydrophilic amino acids.

The ability of the immunophilin FKBP59-HBI to interact with the 90-kDa heat shock protein is encoded by its tetratricopeptide repeat domain.

A protein of apparent molecular mass of approximately 59 kDa of the FK506-binding protein class (FKBP59) has been found associated with the heat shock protein hsp90 included in nontransformed steroid receptor complexes and termed FKBP59-HBI (HBI for Heat shock protein 90 Binding Immunophilin). Further data analysis has revealed that this immunophilin also belongs to the tetratricopeptide repeat family of proteins. In this work, we describe the hsp90-binding domain of FKBP59-HBI. Density gradient centrifugation, gel filtration, and immunoadsorption analyses failed to demonstrate a stable association between FKBP59-HBI and hsp90 in the rabbit reticulocyte lysate. Using a gel-retardation assay, we provide evidence for a specific ATP-independent interaction between highly purified wild-type rabbit FKBP59-HBI and human hsp90 beta. This interaction was not affected by the immunosuppressants FK506 and rapamycin. Examination of the behavior of several mutants led us to conclude that the tetratricopeptide motifs localized in the C-terminal part of FKBP59-HBI are necessary for hsp90 binding.

Changes in protein ubiquitination and the expression of ubiquitin‐encoding transcripts in daylily petals during floral development and senescence

The occurrence of ubiquitin‐protein conjugates and the expression of ubiquitin genes during floral development and senescence of Hemerocallis fulva (daylily) is reported. Daylily petal proteins were extracted at various stages of floral development and the abundance of ubiquitinated proteins determined by western blotting with affinity purified antibody to ubiquitin. Up to 100 ubiquitin conjugates in the molecular mass range 22–220 kDa were detected within the petals during bud development through to flower opening. The intensity of several of the ubiquitinated protein bands changed markedly during flower opening and/or senescence. Ubiquitinated proteins of apparent molecular masses 160, 105 and 29 kDa were intense in the daylily bud, but rapidly disappeared after flower opening, whereas ubiquitinated proteins of apparent molecular masses 26 and 15.5 kDa increased in intensity as the flowers senesced. Treatment of daylily buds with a 1‐h pulse of cycloheximide as the flowers commenced opening prevented these changes; this treatment also delayed the onset of visual signs of senescence by 5–6 days. Analysis by northern blotting demonstrated differential expression of ubiquitin transcripts by the multi‐ubiquitin gene family during floral development and senescence. Inhibition of petal senescence by treatment with cycloheximide resulted in the accumulation of several ubiquitin‐encoding transcripts and the down‐regulation of a 3.4 kb transcript. Overall, these studies suggest that ubiquitin is involved in the degradation of many petal proteins during floral development and senescence and that, during senescence, this selective degradation is occurring against a background of net proteolysis.

Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

Human small intestinal angiotensin-converting enzyme: intracellular transport, secretion and glycosylation.

Human intestinal angiotensin-converting enzyme (ACE) exists in the brush-border membrane as a monomeric protein of apparent molecular mass 184 kDa. It is associated with the membrane via a hydrophobic segment and has a transmembrane orientation [Naim (1992) Biochem. J. 286, 451-457]. In addition to the membrane-bound form (ACEm), hydrophilic forms of ACE (ACEsec) can be identified in biosynthetically labelled intestinal cells. Thus the culture medium of biosynthetically labelled human biopsy samples contains an ACE molecule which has an apparent molecular mass similar to that of its membrane-bound counterpart. The secreted ACEsec forms follow a precursor/product relationship with the mature ACE molecule. The effect of the monomeric structure of ACE in its intracellular transport and secretion was investigated by pulse-chase experiments on human biopsy samples labelled with [35S]methionine. The results reveal 2-3-fold slower transport of ACE from the endoplasmic reticulum (ER) to the Golgi as compared with the homodimeric proteins dipeptidylpeptidase IV and aminopeptidase N. Further, the transport kinetics of ACE are comparable with those of human sucrase-isomaltase and human maltase-glucoamylase, two brush-border disaccharidases that do not form homodimers in the ER of human small-intestinal cells. These findings strongly suggest that homodimerization of brush-border proteins may influence the rate of transport of these proteins from the ER to the Golgi. The effect of glycosylation on the transport and secretion of ACE was investigated by utilizing several inhibitors of glycan processing. Here, secretion of ACE molecules continued to take place, albeit to a considerably lesser extent. In fact, approx. 2-fold less ACE molecules were secreted in the presence of inhibitors of ER glucosidases I and II and cis-Golgi mannosidase-I, suggesting that carbohydrate processing is important in the attainment of a transport-competent conformation.

Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: subcellular distribution of a protein required for yeast protein secretion.

Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37 degrees C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic test confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.