scholarly journals Aminopeptidases trim Xaa-Pro proteins, initiating their degradation by the Pro/N-degron pathway

2021 ◽  
Vol 118 (43) ◽  
pp. e2115430118
Author(s):  
Shun-Jia Chen ◽  
Leehyeon Kim ◽  
Hyun Kyu Song ◽  
Alexander Varshavsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Ubiquitin Ligase ◽  
Proteolytic System ◽  
A Subunit

N-degron pathways are proteolytic systems that recognize proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Our previous work identified Gid4 as a recognition component (N-recognin) of the Saccharomyces cerevisiae proteolytic system termed the proline (Pro)/N-degron pathway. Gid4 is a subunit of the oligomeric glucose-induced degradation (GID) ubiquitin ligase. Gid4 targets proteins through the binding to their Nt-Pro residue. Gid4 is also required for degradation of Nt-Xaa-Pro (Xaa is any amino acid residue) proteins such as Nt-[Ala-Pro]-Aro10 and Nt-[Ser-Pro]-Pck1, with Pro at position 2. Here, we show that specific aminopeptidases function as components of the Pro/N-degron pathway by removing Nt-Ala or Nt-Ser and yielding Nt-Pro, which can be recognized by Gid4-GID. Nt-Ala is removed by the previously uncharacterized aminopeptidase Fra1. The enzymatic activity of Fra1 is shown to be essential for the GID-dependent degradation of Nt-[Ala-Pro]-Aro10. Fra1 can also trim Nt-[Ala-Pro-Pro-Pro] (stopping immediately before the last Pro) and thereby can target for degradation a protein bearing this Nt sequence. Nt-Ser is removed largely by the mitochondrial/cytosolic/nuclear aminopeptidase Icp55. These advances are relevant to eukaryotes from fungi to animals and plants, as Fra1, Icp55, and the GID ubiquitin ligase are conserved in evolution. In addition to discovering the mechanism of targeting of Xaa-Pro proteins, these insights have also expanded the diversity of substrates of the Pro/N-degron pathway.

scholarly journals Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae

1990 ◽  
Vol 268 (2) ◽  
pp. 401-407 ◽  
Author(s):  
A Carabaza ◽  
J Arino ◽  
J W Fox ◽  
C Villar-Palasi ◽  
J J Guinovart
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Glycogen Synthase ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Muscle Enzyme ◽  
Western Blots ◽  
Enzyme Sequence ◽  
A Subunit ◽  
Yeast Glycogen

Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.

Zur Strahleninaktivierung von Ribonuclease

1968 ◽  
Vol 23 (7) ◽  
pp. 934-943 ◽  
Author(s):  
Horst Jung ◽  
Helga Schüssler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Low Temperature ◽  
Gamma Radiation ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Atomic Hydrogen ◽  
Active Components ◽  
Experimental Conditions ◽  
Elution Pattern

Dry ribonuclease was irradiated with 60Co gamma radiation in vacuo, under oxygen atmosphere, and at 77 °K. By chromatography on Sephadex G-50 active ribonuclease was separated from inactive radiation products. From the elution pattern and by ultracentrifugation it was shown that mainly unfolded dimers are formed by gamma irradiation of dry ribonuclease. Amino acid analysis of these various products shows that in all components cystine, methionine, tyrosine, phenylalanine, lysine, and histidine are destroyed with increasing dose whereas glycine shows a small increase. Thus, in ribonuclease irradiated in the dry state the same amino acids are changed as was found after irradiation in aqueous solutions. The radiosensitivity of dry ribonuclease shows an increase by the presence of oxygen of about 2 and a decrease at low temperature in vacuo of about 5. The same factors were also found for the alteration of amino acids, which means that under various experimental conditions amino acid destruction is proportional to loss of enzymatic activity of ribonuclease. The observed selectivity of amino acid destruction may be explained by energy migration or by the attack of atomic hydrogen liberated at random from the molecule. The total number of amino acids destroyed per ribonuclease molecule increases with dose. In enzymatically inactive products this value is always higher by one amino acid residue than in the active components. From this result and from the increase with dose it is concluded that after destruction of one amino acid residue the ribonuclease molecule has a probability (not depending on dose of irradiation) of 0.45 to become inactivated whereas in 55 per cent of all cases the molecule maintains its enzymatic activity.

scholarly journals Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhi-Zhong Song ◽  
Bin Peng ◽  
Zi-Xia Gu ◽  
Mei-Ling Tang ◽  
Bei Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Peach Fruit ◽  
Alcohol Acyltransferase

AbstractThe aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

scholarly journals An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes

Science ◽  
2017 ◽  
Vol 355 (6323) ◽  
pp. eaal3655 ◽  
Author(s):  
Shun-Jia Chen ◽  
Xia Wu ◽  
Brandon Wadas ◽  
Jang-Hyun Oh ◽  
Alexander Varshavsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Sequence Motifs ◽  
Proteolytic System ◽  
Gluconeogenic Enzymes ◽  
Gluconeogenic Enzyme ◽  
Adjacent Sequence

Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. We found that the Gid4 subunit of the ubiquitin ligase GID in the yeast Saccharomyces cerevisiae targeted the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 for degradation. Gid4 recognized the N-terminal proline (Pro) residue and the ~5-residue-long adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2; Gid4 directly or indirectly recognized Pro at position 2 of Pck1, contributing to its targeting. These and related results identified Gid4 as the recognition component of the GID-based proteolytic system termed the Pro/N-end rule pathway. Substrates of this pathway include gluconeogenic enzymes that bear either the N-terminal Pro residue or a Pro at position 2, together with adjacent sequence motifs.

scholarly journals RNA polymerase II mutants defective in transcription of a subset of genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 1010-1016 ◽  
Author(s):  
C Scafe ◽  
M Nonet ◽  
R A Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Amino Acid Residue ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Specific Mrnas ◽  
Type Strains

Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.

scholarly journals Gid10 as an alternative N-recognin of the Pro/N-degron pathway

2019 ◽  
Vol 116 (32) ◽  
pp. 15914-15923 ◽  
Author(s):  
Artem Melnykov ◽  
Shun-Jia Chen ◽  
Alexander Varshavsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Ubiquitin Ligase ◽  
Growth Conditions ◽  
26S Proteasome ◽  
Sequence Motifs ◽  
Yeast Protein ◽  
Substrate Specificities ◽  
Nonfermentable Carbon Source ◽  
A Subunit

In eukaryotes, N-degron pathways (formerly “N-end rule pathways”) comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing degradation of these proteins by the 26S proteasome or autophagy. Gid4, a subunit of the GID ubiquitin ligase in the yeast Saccharomyces cerevisiae, is the recognition component (N-recognin) of the GID-mediated Pro/N-degron pathway. Gid4 targets proteins by recognizing their N-terminal Pro residues or a Pro at position 2, in the presence of distinct adjoining sequence motifs. Under conditions of low or absent glucose, cells make it through gluconeogenesis. When S. cerevisiae grows on a nonfermentable carbon source, its gluconeogenic enzymes Fbp1, Icl1, Mdh2, and Pck1 are expressed and long-lived. Transition to a medium containing glucose inhibits the synthesis of these enzymes and induces their degradation by the Gid4-dependent Pro/N-degron pathway. While studying yeast Gid4, we identified a similar but uncharacterized yeast protein (YGR066C), which we named Gid10. A screen for N-terminal peptide sequences that can bind to Gid10 showed that substrate specificities of Gid10 and Gid4 overlap but are not identical. Gid10 is not expressed under usual (unstressful) growth conditions, but is induced upon starvation or osmotic stresses. Using protein binding analyses and degradation assays with substrates of GID, we show that Gid10 can function as a specific N-recognin of the Pro/N-degron pathway.

RNA polymerase II mutants defective in transcription of a subset of genes

1990 ◽  
Vol 10 (3) ◽  
pp. 1010-1016
Author(s):  
C Scafe ◽  
M Nonet ◽  
R A Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Amino Acid Residue ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Specific Mrnas ◽  
Type Strains

Saccharomyces cerevisiae RNA polymerase II conditional mutants that selectively disrupt the synthesis of specific mRNAs were isolated. At the permissive temperature, several of the mutants were inositol auxotrophs as a result of inadequate induction of INO1 transcription. The transcriptional defects exhibited by one of these Ino- mutants (rpb2-2) were further investigated. The induction of GAL10 and HIS4 transcription in rpb2-2 strains was similar to that of wild-type strains, in contrast to the lack of induction of INO1 transcription. When shifted to the nonpermissive temperature, cells containing rpb2-2 continued to accumulate some mRNAs but not others. Together, these results indicate that transcription of specific genes can be disrupted by RNA polymerase II mutations. The rpb2-2 allele alters an amino acid residue that occurs in a highly conserved segment of the RPB2 protein and that is shared by homologous subunits in other species.

scholarly journals PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (10) ◽  
pp. 5744-5753 ◽  
Author(s):  
C E Brown ◽  
S Z Tarun ◽  
R Boeck ◽  
A B Sachs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
A Subunit ◽  
Two Hybrid

The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails.

scholarly journals Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

2020 ◽  
Vol 117 (25) ◽  
pp. 14158-14167 ◽  
Author(s):  
Cheng Dong ◽  
Shun-Jia Chen ◽  
Artem Melnykov ◽  
Sara Weirich ◽  
Kelly Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Crystal Structures ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Sequence Motifs ◽  
Wild Type ◽  
Rapid Degradation ◽  
A Subunit

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with aKdof 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with aKdof 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeastSaccharomyces cerevisiaecan target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

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