Secretion in yeast: Reconstitution of the translocation and glycosylation of α-factor and invertase in a homologous cell-free system

Rothblatt, Jonathan; Meyer, David I.

doi:10.1016/0092-8674(86)90271-0

Cited by 217 publications

(151 citation statements)

References 46 publications

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“…Invertase (45) and carboxypeptidase Y (50) antisera were prepared as described previously. Anti-a-factor sera was generously provided by J. Rothblatt, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany (42). Rabbit reticulocyte lysate was prepared as described (1).…”

Section: Strains Growth Conditions and Materialsmentioning

confidence: 99%

“…The mechanism of protein permeation across the hydrophobic core of the ER membrane is not understood. Experiments with intermediates artificially blocked at various stages of membrane penetration suggest that this process is mediated by proteins, though they have yet to be identified by the existing assays (14).Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated.…”

mentioning

confidence: 99%

“…Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated.…”

mentioning

confidence: 99%

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A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Deshaies¹,

Schekman²

1987

The Journal of Cell Biology

401

233

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Abstract. We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.T hE first step in the biogenesis of proteins destined for the secretory pathway is their insertion into the membrane of the endoplasmic reticulum (ER).I This process has been studied intensively in mammalian cells through the use of an in vitro assay that faithfully reproduces cotranslational translocation of secretory proteins into the lumen of the ER (2). Dissection of the components required for this activity has revealed the existence of both soluble and membrane-bound factors that participate in protein translocation. The signal recognition particle is a soluble ribonucleoprotein particle consisting of six polypeptides (54) and one molecule of 7SL RNA (55). The signal recognition particle binds to the signal sequence of a nascent preprotein (28, 56), thereby forming a complex that interacts with an integral membrane protein of the ER known as docking protein or signal recognition particle receptor (13,33). This targeting event is followed by cotranslational translocation of the preprotein into the ER lumen. Either during or shortly after the translocation event, the signal sequence is cleaved by the enzyme signal peptidase (10) and core oligosaccharides are transferred to specific asparagine residues (44) of the translocated polypeptide. The mechanism of protein permeation across the hydrophobic core of the ER membrane is not understood. Experiments with intermediates artificially blocked at various stages of membrane penetration suggest that this process is mediated by proteins, though they have yet to be identified by the existing assays (14).Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated. The existence of a posttranslational reaction has allowed these investigators to demonstrate that protein translocation into the yeast ER is energy dependent (15,43,57). In addition, fractionation experiments suggest that the import reaction requires cytosolic components (58). T...

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Section: Strains Growth Conditions and Materialsmentioning

confidence: 99%

mentioning

confidence: 99%

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A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Deshaies¹,

Schekman²

1987

The Journal of Cell Biology

401

233

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“…Preparation of Yeast Microsomes and Analysis of PrP TopologyYeast microsomes were prepared by sucrose gradient centrifugation (38). The membrane topology of PrP was analyzed by a proteinase K (PK) protection assay, performed as described previously for mammalian microsomes (39).…”

Section: Methodsmentioning

confidence: 99%

Cell Surface Expression of the Prion Protein in Yeast Does Not Alter Copper Utilization Phenotypes

Dong

Harris

2004

Journal of Biological Chemistry

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Prion diseases are fatal neurodegenerative disorders that result from conversion of a normal, cell surface glycoprotein (PrP C ) into a conformationally altered isoform (PrP Sc ) that is thought to be infectious. Although a great deal is known about the role of PrP Sc in the disease process, the physiological function of PrP C has remained enigmatic. In this report, we have used the yeast Saccharomyces cerevisiae to test one hypothesized function of PrP C , as a receptor for the uptake or efflux of copper ions. We first modified the PrP signal peptide by replacing its hydrophobic core with the signal sequence from the yeast protein dipeptidyl aminopeptidase B, so that the resulting protein was targeted cotranslationally to the secretory pathway when synthesized in yeast. PrP molecules with the modified signal peptide were efficiently glycosylated, glycolipid-anchored, and localized to the plasma membrane. We then tested whether PrP expression altered the growth deficiency phenotypes of yeast strains harboring deletions in genes that encode key components of copper utilization pathways, including transporters, chaperones, pumps, reductases, and cuproenzymes. We found that PrP did not rescue any of these mutant phenotypes, arguing against a direct role for the protein in copper utilization. Our results provide further clarification of the physiological function of PrP C , and lay the groundwork for using PrPexpressing yeast to study other aspects of prion biology.

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“…Yeast ribosomes were prepared from Saccharomyces cerevisiae strain ABYS 1 by the method of Rothblatt and Meyer [21]. Escherichia coli ribosomes were prepared from strain JM101 as described [22].…”

Section: Methodsmentioning

confidence: 99%

The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

Day

Lord

Roberts

1998

European Journal of Biochemistry

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The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity.Keywords : deoxyribonuclease; ribosome-inactivating protein; RNA N-glycosidase.Ribosome-inactivating proteins (RIPs) are a group of structurally related proteins which depurinate a specific adenyl residue of 28-S ribosomal RNA (A4324 in rat, [1]). Depurination of this residue prevents binding of elongation factors, thereby inhibiting protein synthesis and resulting in cell death. RIPs can be divided into two classes: type I, which consist of a single subunit possessing RNA N-glycosidase activity, and type II, which have a type I-like catalytic subunit (A chain) linked to a cell-binding subunit (B chain). The B chains of plant toxins, e.g. ricin, are typically galactose/N-acetylgalactosamine-specific lectins and are linked to the A chain via a disulphide bond. Bacterial toxins that catalyse the same depurination reaction as RIPs, e.g. Shiga toxin, have a non-covalently bound glycolipidbinding B-chain pentamer.Most type-II RIPs are potent cytotoxins due to their ability to bind to and enter mammalian cells. Once inside the cell, the toxins exploit the cellular retrograde transport system to reach the endoplasmic reticulum from where they translocate into the cytosol by an as yet undetermined mechanism [2]. As holotoxins show no catalytic activity against ribosomes, separation of the A chain and B chain following intoxication is essential in order to exhibit a toxic effect.Type-I RIPs are, by comparison, only weakly cytotoxic when added exogenously to cells. A number of roles have been proposed for type-I RIPs, including defence mechanisms and promotion of senescence. Those which are active on con-specific ribosomes are typically stored in non-cytosolic compartments or as inactive precursors. Such RIPs are believed to be released into the cytosol following cellular damage, such as that caused by viral infection, resulting in local cell suicide around infected areas. An antifungal role has been proposed for RIPs that are inactive against con-specific ribosomes. Barley RIP is co-expressed with a chitinase, and [1, 3] β glucanase, which, while having intrinsic antifungal activity themselves, also facilitate RIP entry i...

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Secretion in yeast: Reconstitution of the translocation and glycosylation of α-factor and invertase in a homologous cell-free system

Cited by 217 publications

References 46 publications

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Cell Surface Expression of the Prion Protein in Yeast Does Not Alter Copper Utilization Phenotypes

The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

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