The structure and function of <i>Saccharomyces cerevisiae</i> proteinase A

Parr, Charity L.; Keates, Robert A. B.; Bryksa, Brian C.; Ogawa, Masahiro; Yada, Rickey Y.

doi:10.1002/yea.1485

Cited by 74 publications

(49 citation statements)

References 64 publications

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“…As shown previously, two bands were present in the Pep4p purification due to autoactivation: the upper one is related to the proteolytic inactive form, and the lower one represents the active form. The Pep4p D294A catalytic dead mutant protease purification showed only the inactive form confirming the loss of proteolytic activity (38). These protease preparations were used in Spt7p cleavage assay, and immunoblot analysis revealed that incubation with the WT Pep4p protease but not with Pep4p D294A or Prc1p results in Spt7p SLIK formation (Fig.…”

Section: Knock-out Screen Reveals That Pep4p Protease Is Required Formentioning

confidence: 67%

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

Spedale

Mischerikow

Heck

et al. 2010

Journal of Biological Chemistry

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The Saccharomyces cerevisiae Spt-Ada-Gcn5 acetyltransferase (SAGA) protein complex is a coactivator for transcription by RNA polymerase II and has various activities, including acetylation and deubuiqitination of histones and recruitment of TATA-binding protein to promoters. The Spt7p subunit is subject to proteolytic cleavage at its C terminus resulting in removal of the Spt8p-binding domain and generation of the SAGA-related SALSA/SAGA-like (SLIK) protein complex. Here, we report identification of the protease responsible for this cleavage. Screening of a protease knock-out collection revealed PEP4 to be required for cleavage of Spt7p within SAGA in vitro. Endogenous formation of truncated Spt7p was abolished in cells lacking PEP4. Purified Pep4p but not catalytic dead mutant Pep4p or unrelated Prc1p protease specifically cleaved Spt7p within SAGA into SLIK-related Spt7p. Interestingly, SAGA lacking Spt8p was more sensitive to Pep4p-mediated truncation of Spt7p, suggesting that Spt8p counteracted its own release from SAGA. Strains mimicking constitutive SLIK formation showed increased resistance to rapamycin treatment, suggesting a role for SLIK in regulating cellular responses to nutrient stress.Transcription initiation by RNA polymerase II is a dynamic process that is regulated by the interplay of a large number of factors. The class of coactivators orchestrates preinitiation complex formation by recruiting nucleosome remodelers, basal transcription factors, and histone modifiers. The evolutionary highly conserved Spt-Ada-Gcn5 acetyltransferase (SAGA)

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Section: Knock-out Screen Reveals That Pep4p Protease Is Required Formentioning

confidence: 67%

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

Spedale

Mischerikow

Heck

et al. 2010

Journal of Biological Chemistry

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“…This suggests that this protein has a function, either for the gongylidia or after ingestion by the ants. Saccharopepsin is known to be important in the activation of yeast vacuolar hydrolases (Parr et al, 2007). This could explain the abundance of this enzyme in the gongylidia as they always have a large vacuole, but it does not explain why this protease is passed on to the fecal fluid.…”

Section: Discussionmentioning

confidence: 98%

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

et al. 2014

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The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5-7, but the aspartic proteases were inactive across a pH range of 3-10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.

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“…the precursors of the Saccharomyces cerevisiae mating factor alpha (MFa) or carboxypeptidase Y (CPY) (Johnson et al, 1987;Julius et al, 1984;Valls et al, 1987;Waters et al, 1988). These pro-peptides have been reported to possess chaperone activity in some cases and are often present to prevent pre-activation of proteins before they reach their final cellular destinations (as is the case for vacuolar proteases, which are produced as inactive pre-forms and become activated by cleavage of their pro-peptides) (Bryant & Stevens, 1998;Klionsky et al, 1990;Parr et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

et al. 2015

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Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

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The structure and function of Saccharomyces cerevisiae proteinase A

Cited by 74 publications

References 64 publications

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

Identification of Pep4p as the Protease Responsible for Formation of the SAGA-related SLIK Protein Complex

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization

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