RESUMO
Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.
Assuntos
Endossomos/metabolismo , Organoides/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Cloreto de Amônio/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Compartimento Celular , Dextranos/metabolismo , Endocitose , Fluoresceína-5-Isotiocianato , Fluoresceínas , Concentração de Íons de Hidrogênio , Cinética , Saccharomyces cerevisiae/ultraestrutura , Espectrometria de Fluorescência , Temperatura , TiocianatosRESUMO
Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae , SolubilidadeRESUMO
Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli beta-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. beta-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150delta, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150delta-beta-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar beta-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.
Assuntos
Citoplasma/enzimologia , Retículo Endoplasmático/metabolismo , Glicoproteínas , Proteínas de Saccharomyces cerevisiae , Leveduras/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Transporte Biológico , Precursores Enzimáticos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Cinética , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo , beta-Lactamases/genéticaRESUMO
Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24 degrees C are preconditioned at 37 degrees C, proteins denatured by subsequent exposure to 48-50 degrees C can be renatured when the cells are allowed to recover at 24 degrees C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24 degrees C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Carboxipeptidases/metabolismo , Catepsina A , Clonagem Molecular , Exocitose , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Temperatura Alta , Glicoproteínas de Membrana/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , beta-Lactamases/metabolismoRESUMO
Rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli beta-lactamase were translocated into the yeast endoplasmic reticulum (ER), glycosylated, misfolded and rapidly degraded. NGFRe underwent ATP-dependent thermosensitive degradation independently of vesicular transport. Since no evidence for degradation by the cytoplasmic 26S proteosome complex could be obtained, NGFRe appeared to be degraded in the ER. Beta-lactamase exited the ER by vesicular traffic and was transported from the Golgi via the Vps10 receptor pathway to the vacuole for degradation. Machineries in the ER and the Golgi appear to recognize distinct structural features on misfolded heterologous proteins and guide them to different degradation pathways.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Saccharomyces cerevisiae/metabolismo , beta-Lactamases/metabolismo , Animais , Escherichia coli/enzimologia , Glicoproteínas/química , Complexo de Golgi/metabolismo , Dobramento de Proteína , Ratos , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética , Vacúolos/metabolismo , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
[3H]alpha-Factor and Lucifer yellow were used to measure receptor mediated and fluid-phase endocytosis in the yeast Saccharomyces cerevisiae, arrested in mitosis by depolymerization of the microtubules or due to a mutation preventing nuclear division (cdc16). Both processes continued at roughly the same level as during interphase. This shows that in yeast endocytosis is not interrupted during mitosis like in mammalian cells.
Assuntos
Endocitose , Mitose , Saccharomyces cerevisiae/metabolismo , Isoquinolinas/metabolismo , Cinética , Fator de Acasalamento , Peptídeos/metabolismo , Feromônios/metabolismo , Saccharomyces cerevisiae/citologiaRESUMO
The N-glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases. Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals. We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver alpha2,3-sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins. The Km values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N. The yeast strain provides an inexpensive self-perpetuating source of ST3N activity for glycan engineering of recombinant proteins.
Assuntos
Parede Celular/enzimologia , Fígado/enzimologia , Polissacarídeos/química , Engenharia de Proteínas , Saccharomyces cerevisiae/química , Sialiltransferases/química , Animais , Assialoglicoproteínas/química , Domínio Catalítico , Cinética , Ácido N-Acetilneuramínico/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Here we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp 150 delta) carrier. The hsp 150 delta-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFRe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp 150 delta-NGFRe fusion protein were highly selectively O-glycosylated.
Assuntos
Glicoproteínas , Manose/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico , Autorradiografia , Sequência de Bases , Western Blotting , Clonagem Molecular , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Glicosilação , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/metabolismo , Metionina/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Conformação Proteica , Ratos , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Radioisótopos de Enxofre , TrítioAssuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Fenômenos Fisiológicos Virais , Parede Celular/metabolismo , Lipossomos , Saccharomyces cerevisiae/ultraestrutura , Esferoplastos , Proteínas do Envelope Viral/metabolismoRESUMO
In mammalian cells intracellular transport is inhibited during mitosis. Here we show that in the yeast Saccharomyces cerevisiae secretion continues uninterrupted during mitosis. S. cerevisiae cells were arrested in mitosis by treating wild-type cells with the microtubule-inhibitor nocodazole, or by incubating a temperature-sensitive cell division cycle mutant (cdc16) at the restrictive temperature. Secretion of invertase into the periplasmic space was equally efficient in mitotic and in unsynchronized cells. Electron microscopy of nocodazole-treated mitotic wild-type cells revealed stretches of rough endoplasmic reticulum, strongly fenestrated Golgi cisternae and clusters of vesicles with the diameter of 30-90 nm. Secretion of invertase was inhibited in mitotic sec7 cells at the restrictive temperature, but continued at the permissive temperature. Sec7 is a mutant strain where intracellular traffic is blocked in unsynchronized cells in the Golgi complex at the restrictive temperature. Thus, the elements of the mitotic Golgi complex appear to be able to support intracellular traffic.
Assuntos
Glicosídeo Hidrolases/metabolismo , Saccharomyces cerevisiae/enzimologia , Benzimidazóis/farmacologia , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Mitose/efeitos dos fármacos , Mutação , Nocodazol , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , beta-FrutofuranosidaseRESUMO
In the preceding paper I reported that Saccharomyces cerevisiae spheroplasts were able to internalize particulate markers, enveloped viruses, into intracellular organelles. Here the internalization of soluble macromolecules into cells having an intact cell wall is described. alpha-Amylase was taken up into cells in a temperature- and concentration-dependent way. The kinetics of accumulation were linear for the first 20-40 min at 37 degrees C and then started to level off. Internalization of alpha-amylase into spheroplasts displayed similar characteristics, but the accumulation rate was about four times higher than into cells. Fluorescent dextran was used to mark morphologically the compartment into which internalization occurred. This marker was accumulated into the vacuole of the cells in a time-, temperature- and concentration-dependent way. A temperature-sensitive mutant deficient in exocytosis was found to be defective in intracellular accumulation of alpha-amylase and dextran. At the restrictive temperature, very little alpha-amylase accumulated into the cells and only faint staining of intracellular organelles with fluorescent dextran could be detected. At the permissive temperatures, accumulation of alpha-amylase and dextran into the mutant cells was comparable with accumulation into wild-type cells. I conclude that alpha-amylase and fluorescent dextran were internalized into S. cerevisiae cells and directed into the vacuoles.
Assuntos
Endocitose , Fluoresceína-5-Isotiocianato/análogos & derivados , Saccharomyces cerevisiae/fisiologia , alfa-Amilases/metabolismo , Dextranos , Fluoresceínas , Cinética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esferoplastos/fisiologia , Vacúolos/fisiologiaRESUMO
When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells.
Assuntos
Endocitose , Saccharomyces cerevisiae/fisiologia , Fracionamento Celular , Membrana Celular/fisiologia , Cinética , Receptores de Concanavalina A/análise , Esferoplastos/fisiologia , Esferoplastos/ultraestrutura , Vírus da Estomatite Vesicular Indiana , VírionRESUMO
Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown. When Saccharomyces cerevisiae cells are pre-conditioned at 37 degrees C, they survive an otherwise lethal exposure to 48-50 degrees C, and form colonies at 24 degrees C. We show here that incubation of yeast cells at 48-50 degrees C, after pre-conditioning at 37 degrees C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER). Soon after return of the cells to 24 degrees C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory beta-lactamase fusion protein remained in the ER for different times. Thereafter their transport competence was resumed very slowly with widely varying kinetics. While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24 degrees C, folded normally, by-passed the heat-affected copies and exited rapidly the ER. The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins. ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Conformação Proteica , Saccharomyces cerevisiae/fisiologia , Adaptação Biológica , Retículo Endoplasmático/fisiologia , Proteínas Fúngicas/ultraestrutura , TemperaturaRESUMO
Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Delta erg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Delta erg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Delta erg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7-1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Glicoproteínas , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Animais , Transporte Biológico Ativo , Membrana Celular/metabolismo , Detergentes , Ergosterol , Genes Fúngicos , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Biológicos , Mutação , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Sialiltransferases/genética , Sialiltransferases/metabolismo , Solubilidade , Vacúolos/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-alpha-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.
Assuntos
Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas , Proteínas de Choque Térmico HSP70 , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Carboxipeptidases/metabolismo , Catepsina A , Ditiotreitol/farmacologia , Glicosídeo Hidrolases/metabolismo , Proteínas de Choque Térmico/metabolismo , Fator de Acasalamento , Oxirredução , Peptídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , beta-FrutofuranosidaseRESUMO
We have studied the relationship between folding and secretion competence of hsp150-beta lactamase fusion proteins in Saccharomyces cerevisiae. hsp150 is a secretory protein of yeast, and beta-lactamase was chosen, since its folding can be monitored by assaying its enzymatic activity. The hsp150 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus. Fusion of beta-lactamase to the C terminus of hsp150 produced Cla-bla protein, which was secretion-competent but inactive. The Pst-bla protein, where beta-lactamase was fused to subunit I, was also inactive and mostly secreted, but part of it remained in the pre-Golgi compartment. When beta-lactamase was fused to the C-terminus of the repetitive region, the fusion protein (Kpn-bla) was translocated to the endoplasmic reticulum, acquired disulfide bonds, and adopted an enzymatically active conformation. Kpn-bla was secreted to the medium without decrease of specific activity or retention in the cell. Folding of Kpn-bla to an active and transport-competent form required co-translational disulfide formation, since treatment of cells with dithiothreitol resulted in endoplasmic reticulum-retained inactive Kpn-bla. When dithiothreitol was removed, Kpn-bla resumed transport competence but remained inactive. Reduction of prefolded Kpn-bla did not inhibit enzymatic activity or transport. The repetitive hsp150 carrier may have use in heterologous protein production by conferring secretion competence to foreign proteins in yeast.