Continued functioning of the secretory pathway is essential for ribosome synthesis

To explore the regulatory elements that maintain the balanced synthesis of the components of the ribosome, we isolated a temperature-sensitive (ts) mutant of Saccharomyces cerevisiae in which transcription both of rRNA and of ribosomal protein genes is defective at the nonpermissive temperature. Temperature sensitivity for growth is recessive and segregates 2:2. A gene that complements the ts phenotype was cloned from a genomic DNA library. Sequence analysis revealed that this gene is SLY1, encoding a protein essential for protein and vesicle transport between the endoplasmic reticulum and the Golgi apparatus. In the strain carrying our ts allele of SLY1, accumulation of the carboxypeptidase Y precursor was detected at the nonpermissive temperature, indicating that the secretory pathway is defective. To ask whether the effect of the ts allele on ribosome synthesis was specific for sly1 or was a general result of the inactivation of the secretion pathway, we assayed the levels of mRNA for several ribosomal proteins in cells carrying ts alleles of sec1, sec7, sec11, sec14, sec18, sec53, or sec63, representing all stages of secretion. In each case, the mRNA levels were severely depressed, suggesting that this is a common feature in mutants of protein secretion. For the mutants tested, transcription of rRNA was also substantially reduced. Furthermore, treatment of a sensitive strain with brefeldin A at a concentration sufficient to block the secretion pathway also led to a decrease of the level of ribosomal protein mRNA, with kinetics suggesting that the effect of a secretion defect is manifest within 15 to 30 min. We conclude that the continued function of the entire secretion pathway is essential for the maintenance of ribosome synthesis. The apparent coupling of membrane synthesis and ribosome synthesis suggest the existence of a regulatory network that connects the production of the various structural elements of the cell.

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Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.2.10.1199 ◽

1982 ◽

Vol 2 (10) ◽

pp. 1199-1204 ◽

Cited By ~ 11

Author(s):

E Kraig ◽

J E Haber ◽

M Rosbash

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Relative Rate ◽

Synthesis Rate ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Mrna Metabolism

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.

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Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.2.10.1199-1204.1982 ◽

1982 ◽

Vol 2 (10) ◽

pp. 1199-1204

Author(s):

E Kraig ◽

J E Haber ◽

M Rosbash

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Relative Rate ◽

Synthesis Rate ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Mrna Metabolism

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.

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Retrograde Transport of Golgi-localized Proteins to the ER

The Journal of Cell Biology ◽

10.1083/jcb.140.1.1 ◽

1998 ◽

Vol 140 (1) ◽

pp. 1-15 ◽

Cited By ~ 153

Author(s):

Nelson B. Cole ◽

Jan Ellenberg ◽

Jia Song ◽

Diane DiEuliis ◽

Jennifer Lippincott-Schwartz

Keyword(s):

Plasma Membrane ◽

Golgi Complex ◽

Fusion Proteins ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Viral Glycoprotein ◽

Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

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New Mutants of Saccharomyces cerevisiae Affected in the Transport of Proteins From the Endoplasmic Reticulum to the Golgi Complex

Genetics ◽

10.1093/genetics/142.2.393 ◽

1996 ◽

Vol 142 (2) ◽

pp. 393-406 ◽

Cited By ~ 11

Author(s):

Linda J Wuestehube ◽

Rainer Duden ◽

Arlene Eun ◽

Susan Hamamoto ◽

Paul Korn ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Secretory Protein ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Membrane Structures ◽

Α Subunit ◽

Complementation Groups ◽

Vacuolar Protein

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

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Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

10.1101/2021.10.05.463164 ◽

2021 ◽

Author(s):

Benjamin Pillet ◽

Alfonso Méndez-Godoy ◽

Guillaume Murat ◽

Sébastien Favre ◽

Michael Stumpe ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Translational Regulation ◽

De Novo ◽

Spatial Separation ◽

Ribosome Assembly ◽

Mrna Levels ◽

Ribosomal Assembly ◽

Surplus Production ◽

Regulatory Machinery

AbstractThe biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

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P53 is Activated without RPL11 Upregulation in Diamond-Blackfan Anemia,

Blood ◽

10.1182/blood.v118.21.3432.3432 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3432-3432

Author(s):

Hong-Yan Du ◽

M. Tarek Elghetany ◽

Blanche P Alter ◽

Akiko Shimamura

Keyword(s):

Bone Marrow ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Mrna Levels ◽

Protein Levels ◽

Diamond Blackfan Anemia ◽

Human Cd34 ◽

Cd34 Cells ◽

P53 Activation ◽

Ribosomal Stress

Abstract Abstract 3432 Diamond-Blackfan anemia (DBA) is an autosomal dominantly inherited bone marrow failure syndrome characterized by red cell aplasia, physical anomalies, and cancer predisposition. DBA is caused by mutations resulting in haploinsufficiency of genes encoding ribosomal proteins. p53 is activated in the erythroid lineage following reduction of ribosomal protein expression; however the mechanism whereby ribosomal stress results in p53 activation in DBA remains unclear. RPL11 has been proposed to play a central role in p53 activation following ribosomal stress. Reduced expression of individual small ribosomal subunit proteins in a tumor cell line resulted in increased translation of RPL11. Excess free RPL11 can bind and inactivate HDM2, an E3 ubiquitin ligase targeting p53 for degradation. The recent demonstration that cellular responses to ribosomal perturbations vary widely between different tissues raised the question of whether RPL11 upregulation contributes to p53 activation following ribosomal stress in hematopoietic progenitors. To address this question, we modeled DBA in human CD34+ cells. Since RPS19 is the most commonly mutated gene in DBA, we used lentiviral vectors expressing short hairpin RNAs to knock down RPS19 expression in primary human CD34+ cells. RPS19 protein levels were reduced to about 50% of control levels in a manner reflecting the haploinsufficient state in DBA. RPS19 depletion resulted in elevated p53 protein levels and increased mRNA levels of p21, a transcriptional target of p53. Total p53 mRNA levels and p53 mRNA translational activity remained unchanged consistent with a post-transcriptional mechanism for p53 activation. Although total RPL11 mRNA levels were not diminished following RPS19 depletion, RPL11 protein levels were significantly decreased consistent with post-transcriptional downregulation. Depletion of RPS19 in human CD34+ cells did not affect polysome loading of RPL11 mRNA. Reduction of additional ribosomal proteins also accompanied RPS19 knockdown consistent with coordinate regulation of multiple ribosomal protein levels. Corticosteroids, which improve anemia in the majority of DBA patients, did not prevent p53 activation, nor did this improve RPS19 or RPL11 protein levels. Expression of p53 was also assessed in bone marrow biopsy slides from 26 DBA patients with the following genotypes: RPS19 (18), RPS24 (2), RPS26 (2), RPS10 (1), RPS17 (1), RPS7 (1), and RPL11 (1). p53 was over-expressed in all but one patient (RPS26), and was clearly over-expressed in the DBA patient harboring the RPL11 mutation. In summary, we find that p53 activation in DBA does not require upregulation of RPL11 translation or elevated RPL11 protein levels. p53 activation persists in DBA caused by RPL11 deficiency. Corticosteroids do not improve ribosomal protein levels nor do they prevent p53 activation. Disclosures: No relevant conflicts of interest to declare.

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Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5393 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5393-5404 ◽

Cited By ~ 73

Author(s):

Baojie Li ◽

Concepcion R. Nierras ◽

Jonathan R. Warner

Keyword(s):

Rna Polymerase Ii ◽

Mating Type ◽

Binding Sites ◽

Transcription Initiation ◽

Ribosomal Proteins ◽

Secretory Pathway ◽

Rrna Genes ◽

Mrna Levels ◽

Growing Cell ◽

Mating Type Genes

ABSTRACT The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 orGAL1 demonstrated that the Rap1p binding sites ofRPL30 are sufficient to silence the transcription ofACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.

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SSB, Encoding a Ribosome-Associated Chaperone, Is Coordinately Regulated with Ribosomal Protein Genes

Journal of Bacteriology ◽

10.1128/jb.181.10.3136-3143.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3136-3143 ◽

Cited By ~ 31

Author(s):

Nelson Lopez ◽

John Halladay ◽

William Walter ◽

Elizabeth A. Craig

Keyword(s):

Heat Shock ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Chain Complex ◽

Mrna Levels ◽

Ribosomal Protein Genes ◽

Genes Encoding ◽

Synthetic Capacity ◽

Translational Apparatus ◽

Temperature Upshift

ABSTRACT Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1, of the gene encoding the heat shock factor (HSF). However, upon a heat shock theEXA3-1 mutation did not significantly alter the reduction in the mRNA levels of two genes encoding proteins unrelated to the translational apparatus. Analysis of gene fusions indicated that the transcribed region, but not the promoter of SSB, is sufficient for this HSF-dependent regulation. Our studies suggest that Ssb is regulated like a core component of the ribosome and that HSF is required for proper regulation of SSB and ribosomal mRNA after a temperature upshift.

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BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast.

The Journal of Cell Biology ◽

10.1083/jcb.130.1.41 ◽

1995 ◽

Vol 130 (1) ◽

pp. 41-49 ◽

Cited By ~ 133

Author(s):

J F Simons ◽

S Ferro-Novick ◽

M D Rose ◽

A Helenius

Keyword(s):

Molecular Chaperone ◽

Intracellular Transport ◽

Critical Role ◽

Carboxypeptidase Y ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Wild Type ◽

Yeast Strains ◽

Mutant Strains

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER-retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.

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