Secretion in yeast: translocation and glycosylation of prepro-α-factor <i>in vitro</i>
 can occur via an ATP-dependent post-translational mechanism

Rothblatt, Jonathan; Meyer, David I.

doi:10.1002/j.1460-2075.1986.tb04318.x

Cited by 184 publications

(119 citation statements)

References 14 publications

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“…5C). In line with our data, pp␣F was previously shown to be also post-translationally translocated into yeast microsomes when synthesized in a yeast cell-free or a rabbit reticulocyte lysate translation system (49).…”

Section: Discussionsupporting

confidence: 91%

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

Loibl

Wunderle

Hutzler

et al. 2014

Journal of Biological Chemistry

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Background: O-Mannosylation is a conserved, essential protein modification that is initiated in the endoplasmic reticulum (ER). Results: Protein O-mannosyltransferases act at the translocon complex to modify proteins entering the ER. Conclusion: Protein translocation into the ER, O-mannosylation, and N-glycosylation are coordinated processes. Significance: Defining the mechanism of O-mannosylation is crucial to understand its diverse physiological roles for ER homeostasis.

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Section: Discussionsupporting

confidence: 91%

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

Loibl

Wunderle

Hutzler

et al. 2014

Journal of Biological Chemistry

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“…Moreover, the translocation process was abolished after enzymatic ATP degradation by apyrase and could be restored by addition of ATP or more effectively by creatine phosphate. Although the true nature of the energy source cannot be decided from our preliminary results, they are in agreement with data from other laboratories showing that energy-rich phosphate bonds are used for transport across the endoplasmic reticulum [8,9,11,261. This differs from post-translational translocation into mitochondria [27,281 or protein transport across the prokaryotic plasma membrane [29], where both a membrane potential and ATP are required.…”

Section: Discussionsupporting

confidence: 91%

“…This could mean that the difference reflects an intrinsic feature of a-factor or that the yeast system has special properties. It should be mentioned that post-translational import of prepro-a-factor in a heterologous system, using canine-pancreatic microsomes, did not work [7,9]. Studies by Zimmermann and coworkers have demonstrated for several small proteins a post-translational translocation of completed peptide chains and postulate an upper size limit of about 9 kDa to maintain an insertion-competent conformation without the aid of a signal-recognition protein (SRP) and docking protein.…”

Section: Discussionmentioning

confidence: 99%

Post‐translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent

Roitsch

Lehle

1988

European Journal of Biochemistry

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The translation and translocation of two yeast glycoproteins, invertase and carboxypeptidase Y, were studied in a heterologous cell-free translation system from reticulocytes supplemented with dog pancreas microsomes. Using in vitro synthesized mRNA transcripts, encoding complete or truncated invertase forms, the influence of polypeptide size and ribosome dependence was studied. It was found that C-terminal truncated fragments of 25 kDa, i.e. a size larger than the average size of a domain structure, are translocated and processed posttranslationally with a similar efficiency to the cotranslational events. Post-translational import decreases with increasing peptide chain, mature polypeptide (60 kDa) being no longer translocated. Post-translational competence is only maintained as long as the peptide remains associated with ribosomes. Translocation of invertase depends on the presence of the leader peptide and requires energy independent of protein synthesis. Size dependence of post-translational import could also be demonstrated for carboxypeptidase Y .The decisive initial step in the process of secretion of eukaryotic proteins is the translocation of the nascent polypeptide chain across the rough endoplasmic reticulum membrane (for review see [I -31). Through the use of cell-free protein-translation systems much has been learned about the molecular mechanism of this process. Until recently it was thought that this receptor-mediated process, as proposed in the so-called signal hypothesis [4,5], is a cotranslational event, i.e. protein synthesis, membrane recognition and insertion into or transfer across the membrane are tightly coupled. In this way it was thought to differ from secretion of nuclearencoded proteins into mitochondria, chloroplasts or peroxisomes, which occurs post-translationally. However, recent in vitro studies from several groups indicate that for certain small proteins also a post-translational energy-dependent translocation across the endoplasmic reticulum can be observed (for review see Plasmid constructionsYEpl3-SUC2 contains a 5.1-kb yeast genomic DNA fragment which includes the SUC2 gene encoding invertase. The coding region for cytoplasmic invertase was subcloned from this as a 2090-bp HindIII fragment into the HindIII site of a pGEM2 transcription vector (Genofit) resulting in plasmid pRT18.39. Clones with the right orientation of the insert were identified by restriction analysis.The coding region for the precursor form of the secretory invertase was also subcloned from YEpl3-SUC2. A fragment from a limited HindIII and complete AccI digestion was purified from a preparative low-melting-point agarose gel and ligated into the HindIIIIAccI cloning site of pGEM2 giving rise to pRT23.04.To delete about half of the coding region of the SUC2 gene, while preserving the N-terminal and C-terminal sequences,

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“…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).…”

mentioning

confidence: 99%

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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Secretion in yeast: translocation and glycosylation of prepro-α-factor in vitro can occur via an ATP-dependent post-translational mechanism

Cited by 184 publications

References 14 publications

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

Post‐translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent

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

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