Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “secretion defect”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed1990

Selective enrichment for temperature-sensitive secretion mutants of mammalian cells using plant lectin, concanavalin A.

The use of conditional mutants as a genetic approach to study protein secretion in mammalian cells requires the isolation of a large number of mutants. Because a procedure for the direct selection of mutants with secretion defects is not available, their isolation depends upon the selective enrichment of mutant phenotypes in a cell population. We have devised an enrichment strategy in which rat hepatoma cells unable to replace surface membrane receptors of a plant lectin, concanavalin A, are resistant to the cytotoxic effects of this lectin when administered at a nonpermissive temperature. This treatment yields a population highly enriched in cells that demonstrate temperature-sensitive secretion. Therefore, this selection strategy has important application in isolating temperature-sensitive mutants for use in the study of the mammalian cell secretion pathway.

Animals↗
PubMed1979

Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells contain a small internal pool of the secretory enzymes invertase and acid phosphatase. This pool increases up to 8-fold at 37 degrees C in a temperature-sensitive, secretion-defective mutant strain (sec 1-1). Cell division and incorporation of a sulfate permease activity stop abruptly at the restrictive temperature, while protein synthesis continues for several hours. Electron microscopy of mutant cells incubated at 37 degrees C reveals a large increase in the number of intracellular membrane-bound vesicles, which are shown by histochemical staining to contain the accumulated acid phosphatase. The vesicles are removed and the accumulated enzymes are secreted when cells are returned to a permissive temperature in the presence or absence of cycloheximide. These results are consistent with a vesicle intermediate in the yeast secretory pathway and suggest that exocytosis may contribute to cell-surface growth.

Acid Phosphatase↗
PubMed2000

A novel von Willebrand disease-causing mutation (Arg273Trp) in the von Willebrand factor propeptide that results in defective multimerization and secretion.

In this report we describe the molecular defect underlying partial and severe quantitative von Willebrand factor (VWF) deficiencies in 3 families previously diagnosed with types 1 and 3 Von Willebrand-disease. Analysis of the VWF gene in affected family members revealed a novel C to T transition at nucleotide 1067 of the VWF complemetary DNA (cDNA), predicting substitution of arginine by tryptophan at amino acid position 273 (R273W) of pre-pro-VWF. Two patients, homozygous for the R273W mutation, had a partial VWF deficiency (VWF:Ag levels of 0.06 IU/mL and 0.09 IU/mL) and lacked high-molecular weight VWF multimers in plasma. A third patient, also homozygous for the R273W mutation, had a severe VWF deficiency (VWF:Ag level of less than 0.01 IU/mL) and undetectable VWF multimers in plasma. Recombinant VWF having the R273W mutation was expressed in COS-7 cells. Pulse-chase experiments showed that secretion of rVWFR273W was severely impaired compared with wild-type rVWF. However, the mutation did not affect the ability of VWF to form dimers in the endoplasmic reticulum (ER). Multimer analysis showed that rVWFR273W failed to form high-molecular-weight multimers present in wild-type rVWF. We concluded that the R273W mutation is responsible for the quantitative VWF deficiencies and aberrant multimer patterns observed in the affected family members. To identify factors that may function in the intracellular retention of rVWFR273W, we investigated the interactions of VWF expressed in COS-7 cells with molecular chaperones of the ER. The R273W mutation did not affect the ability of VWF to bind to BiP, Grp94, ERp72, calnexin, and calreticulin in COS-7 cells. (Blood. 2000;96:560-568)

Amino Acid Sequence↗
PubMed2003

Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming.

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.

Bacterial Outer Membrane Proteins↗
PubMed1996

The mutation Gly142-->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes.

While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study are involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142e hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL.

Animals↗
PubMed2004

A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency.

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5.0% and 7.3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild-type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 A of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis-folded domain, leading to defective secretion.

Adult↗
PubMed1983

Altered arachidonic acid synthesis and lipid peroxidation in diabetes mellitus: possible roles in leukocyte dysfunction and other cellular defects.

Hyperglycemic diabetics are prone to unusual or especially severe infections; at the cellular level, diabetic polymorphonuclear leukocytes (PMNs) show defects in several antimicrobial functions. However, the basis for these defects is unknown, and they may not be fully ascribable to hyperglycemia, hypoinsulinemia or acidosis alone. Recently, it has been shown that several important PMN functions may be mediated (at least in part) by metabolites of arachidonic acid synthesized via the lipoxygenase pathway, especially arachidonate hydroperoxides and leukotriene (LT) B4. We speculate that synthesis of these mediators may be deficient in severely hyperglycemic diabetics (fasting plasma glucose greater than 250-300 mg/dl) due to deficiencies of substrate (arachidonic acid) synthesis and release. Such defects might be expected since, in animal studies, severe insulin lack and glucagon excess inhibit the desaturation of precursor fatty acids to arachidonic acid. On the other hand, whereas low levels of lipid peroxides or their derivatives may be required in certain cells for normal function, excessive levels of such compounds also are detrimental to cellular function and could play a role as well in the complications of milder or partially treated diabetics who manifest high basal insulin levels. For example, cells which may be particularly sensitive to an excess of peroxides include islet beta cells, PMNs and possibly vascular endothelial cells (all of which appear to be deficient in glutathione peroxidase). These observations suggest a role for accumulation of lipid peroxides in the impaired insulin secretion, defective PMN function and possibly endothelial death and increased vascular (retinal, endothelial, and renal) permeability of some milder diabetics. The available data are compatible with the speculation that in partially treated or lesser degrees of hyperglycemia, increased arachidonate synthesis and excessive lipid peroxidation may be present. Although it remains to be established that all of the results from experimentally-induced diabetics can be extrapolated to humans, these findings suggest that the cell damage attendant upon peroxide generation might be susceptible to prophylactic treatment with anti-oxidants such as alpha-tocopherol or ascorbic acid. In the more severe or later stages of hyperglycemia, a deficiency of lipoxygenase-derived products may supervene; dietary modifications designed to increase essential fatty acid availability might present a unique ancillary therapeutic approach at this stage of diabetes.

Arachidonic Acid↗
PubMed1995

Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

Amino Acid Sequence↗
PubMed2005

Hyper-IgE syndrome is not associated with defects in several candidate toll-like receptor pathway genes.

The genetic basis of hyper-IgE syndrome (HIES), also known as Job syndrome, a primary immunodeficiency associated with recurrent skin and pulmonary infections, is unknown. We hypothesized that HIES is due to a defect in the Toll-like receptor signaling pathway. We used a whole blood cytokine assay to compare inflammatory responses to stimulation with specific Toll-like receptor (TLR) pathway agonists in four individuals with HIES and nine healthy controls. Production of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-12 was not impaired in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, lipoteichoic acid, Staphylococcus aureus, Escherichia coli, or Streptococcus pneumoniae. Interferon (IFN)-gamma was reduced in HIES subjects in response to each of these stimuli. We sequenced several candidate genes from the TLR pathway in HIES individuals to determine whether any mutations were associated with this syndrome. No novel mutations or polymorphisms were found in the coding regions of TLR1, TLR2, TLR6, MyD88, or TRAF6. In summary, although HIES individuals had an IFN-gamma secretion defect, they also produced normal levels of several TLR-regulated proinflammatory cytokines. No unique mutations or polymorphisms were observed in several candidate genes from the TLR pathway. Our studies do not support a role for a defective TLR response in HIES individuals.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Apolipoprotein B48 glycosylation in abetalipoproteinemia and Anderson's disease.

BACKGROUND & AIMS: Abetalipoproteinemia and Anderson's disease are hereditary lipid malabsorption syndromes. In abetalipoproteinemia, lipoprotein assembly is defective because of mutations in the microsomal triglyceride transfer protein. Here, we evaluated the intracellular transport of apolipoprotein B48 to localize the defect in Anderson's disease. METHODS: Asparagine-linked oligosaccharide processing of apolipoprotein B48 in normal and affected individuals was determined by the endoglycosidase H and F sensitivities of the protein after metabolic labeling of intestinal explants in organ culture. Cell ultrastructure was evaluated with electron microscopy. RESULTS: In Anderson's disease as in normal individuals, there was a time-dependent transformation of high mannose endoglycosidase H-sensitive oligosaccharides, of endoplasmic reticulum origin, to complex endoglycosidase H-resistant oligosaccharides, added in the Golgi network. In contrast, despite the translocation of apolipoprotein B48 into the endoplasmic reticulum in patients with abetalipoproteinemia and in biopsies treated with Brefeldin A, which blocks anterograde transport between the endoplasmic reticulum and the Golgi network, there was no transformation of endoglycosidase H-sensitive oligosaccharides. CONCLUSIONS: In abetalipoproteinemia and Anderson's disease, apolipoprotein B48 is completely translocated into the endoplasmic reticulum, but only in Anderson's disease is the protein transported to the Golgi apparatus. This suggests that Anderson's disease is caused by a post-Golgi cargo-specific secretion defect.

Abetalipoproteinemia↗
PubMed2006

Foxa1-deficient mice exhibit impaired insulin secretion due to uncoupled oxidative phosphorylation.

Foxa1 (formerly hepatic nuclear factor 3alpha) belongs to the family of Foxa genes that are expressed in early development and takes part in the differentiation of endoderm-derived organs and the regulation of glucose homeostasis. Foxa1-/- pups are growth retarded and hypoglycemic but glucose intolerant in response to an intraperitoneal glucose challenge. However, the mechanism of glucose intolerance in this model has not been investigated. Here, we show that Foxa1-/- islets exhibit decreased glucose-stimulated insulin release in islet perifusion experiments and have significantly reduced pancreatic insulin and glucagon content. Moreover, Foxa1-/- beta-cells exhibit attenuated calcium influx in response to glucose and glyburide, suggesting an insulin secretion defect either at the level or upstream of the ATP-sensitive K+ channel. Intracellular ATP levels after incubation with 10 mmol/l glucose were about 2.5 times lower in Foxa1-/- islets compared with controls. This diminished ATP synthesis could be explained by increased expression of the mitochondrial uncoupling protein uncoupling protein 2 (UCP2) in Foxa1-deficient islets, resulting in partially uncoupled mitochondria. Chromatin immunoprecipitation assays indicate that UCP2 is a direct transcriptional target of Foxa1 in vivo. Thus, we have identified a novel function for Foxa1 in the regulation of oxidative phosphorylation in pancreatic beta-cells.

Animals↗
PubMed1978

Evidence for a defect in pituitary secretion of luteinizing hormone in chronic alcoholic men.

To characterize the defect in the hypothalamic-pituitary-gonadal axis of alcoholic men, acute and chronic LRF responses were evaluated in 22 chronic alcoholic men with varying degrees of biochemically and histologically confirmed liver disease. In addition, acute LRF responses in 14 normal men, before and at the end of 72 h of administration of 2 ml/kg/day 95% ethanol, were evaluated. The alcoholics hd significantly reduced basal testosterone and elevated gonadotropin levels (both FSH and LH) compared to the normal volunteers (P less than 0.02). Serum concentrations of estradiol and PRL did not differ between alcoholics and normal volunteers. A 100-micrograms bolus of LRF resulted in a 3-fold increase of LH in alcoholic men as compared to a 6-fold increase of serum LH in normal volunteers. No significant difference in the LRF-induced FSH responses was observed. When the response of normal volunteers to LRF before and after ethanol administration was evaluated, basal levels of both gonadotropins were increased after alcohol administration and a reduced LRF-induced LH response was observed. Based upon these results, we conclude that: 1) the central hypothalamic-pituitary defect known to exist for LH secretion is in part due to inadequate pituitary secretion and 2) acute alcohol ingestion in normal men suppresses the LRF-induced LH but not the FSH response.

Adult↗
PubMed2001

Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism.

Mutations in hepatocyte nuclear factor 1alpha (HNF-1alpha) lead to maturity-onset diabetes of the young type 3 as a result of impaired insulin secretory response in pancreatic beta-cells. The expression of 50 genes essential for normal beta-cell function was studied to better define the molecular mechanism underlying the insulin secretion defect in Hnf-1alpha(-/-) mice. We found decreased steady-state mRNA levels of genes encoding glucose transporter 2 (Glut2), neutral and basic amino acid transporter, liver pyruvate kinase (L-Pk), and insulin in Hnf-1alpha(-/-) mice. In addition, we determined that the expression of several islet-enriched transcription factors, including Pdx-1, Hnf-4alpha, and Neuro-D1/Beta-2, was reduced in Hnf-1alpha(-/-) mice. These changes in pancreatic islet mRNA levels were already apparent in newborn animals, suggesting that loss of Hnf-1alpha function rather than chronic hyperglycemia is the primary cause of the altered gene expression. This expression profile was pancreatic islet-specific and distinct from hepatocytes, where we found normal expression of Glut2, L-Pk, and Hnf-4alpha in the liver of Hnf-1alpha(-/-) mice. The expression of small heterodimer partner (Shp-1), an orphan receptor that can heterodimerize with Hnf-4alpha and inhibit its transcriptional activity, was also reduced in Hnf-1alpha(-/-) islets. We characterized a 0.58-kb Shp-1 promoter and determined that the decreased expression of Shp-1 may be indirectly mediated by a downregulation of Hnf-4alpha. We further showed that Shp-1 can repress its own transcriptional activation by inhibiting Hnf-4alpha function, thereby establishing a feedback autoregulatory loop. Our results indicate that loss of Hnf-1alpha function leads to altered expression of genes involved in glucose-stimulated insulin secretion, insulin synthesis, and beta-cell differentiation.

Animals↗
PubMed1988

Synthesis, processing, and transport of Pseudomonas aeruginosa elastase.

Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and Safrin, J. Bacteriol. 170:1215-1219, 1988), forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1996

Increased insulin resistance and insulin secretion in nondiabetic African-Americans and Hispanics compared with non-Hispanic whites. The Insulin Resistance Atherosclerosis Study.

The etiology of NIDDM is still controversial, with both insulin resistance and decreased insulin secretion postulated as potential important factors. African-Americans and Hispanics have a two- to threefold excess risk of developing NIDDM compared with non-Hispanic whites. Yet little is known concerning the prevalence of insulin resistance and secretion defects in minorities, especially in African-Americans in population-based studies. Fasting and 2-h post-glucose load glucose and insulin levels, insulin-mediated glucose disposal (insulin sensitivity index) (S(I)), glucose effectiveness (S(G)), and first-phase insulin response (acute insulin response [AIR]) were determined in nondiabetic African-Americans (n= 288), Hispanics (n= 363), and non-Hispanic whites (n= 435) as part of the Insulin Resistance Atherosclerosis Study. Subjects received a standard 2-h oral glucose tolerance test on the first day and an insulin-modified frequently sampled intravenous glucose tolerance test on the second day. African-Americans and Hispanics were more obese than non-Hispanic whites. Both African-Americans and Hispanics had higher fasting and 2-h insulin concentrations and AIR but lower S(I) than non-Hispanic whites. No ethnic difference was observed in S(G). After further adjustments for obesity, body fat distribution, and behavioral factors, African-Americans continued to have higher fasting and 2-h insulin levels and AIR, but lower S(I) than non-Hispanic whites. In contrast, after adjustment for these covariates, no significant ethnic differences in S(I) or fasting insulin levels were observed between Hispanics and non-Hispanic whites. Hispanics continued to have higher 2-h insulin levels and AIRs than those in non-Hispanic whites. In this report, the association between S(I) and upper body adiposity (waist-to-hip, ratio) was similar in each ethnic group. Both nondiabetic African-Americans and Hispanics have increased insulin resistance and higher AIR than nondiabetic non-Hispanic whites, suggesting that greater insulin resistance may be in large part responsible for the higher prevalence of NIDDM in these minority groups. However, in Hispanics. the greater insulin resistance may be due to greater adiposity and other behavioral factors.

Africa↗
PubMed1996

Yeast protein translocation complex: isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61 beta subunit.

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion-defective sec15-1 mutation. This gene encodes a protein homologous to the beta subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane-spanning region at the C-terminus but no N-terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61 beta subunit and 34% identity to the Arabidopsis thaliana Sec61 beta subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61-2 and sec61-3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N-terminus exposed to the cytoplasm. The in vitro translated Seb1p was post-translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature-sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis.

Amino Acid Sequence↗
PubMed1998

Genetic interaction between YPT6 and YPT1 in Saccharomyces cerevisiae.

Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures. Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both. Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain. SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain. Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele. These data suggest that Ypt6p has some redundant function with Ypt1p. However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p. This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway.

Biological Transport↗
PubMed2000

Differential fates of invertase mutants in the yeast endoplasmic reticulum.

A number of proteins have been identified as substrates for endoplasmic reticulum (ER)-associated protein degradation (ERAD) and we describe here a new model substrate with which to study this process. Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538p levels were low, while Suc2-533p was present in high amounts. Because Suc2-533p and Suc2-538p mRNA levels were comparable, we examined whether Suc2-538p was targeted for degradation. Both mutant polypeptide levels were unaffected in a yeast strain deficient in vacuolar protease activity and, additionally, we showed that Suc2-538p was stabilized in ERAD-deficient strains, demonstrating that Suc2-538p was a substrate for ERAD.

Animals↗