Review: Biosynthesis and function of yeast vacuolar proteases

Hazel, H. Bart van den; Kielland‐Brandt, Morten C.; Winther, Jakob R.

doi:10.1002/(sici)1097-0061(199601)12:1<1::aid-yea902>3.0.co;2-n

Cited by 177 publications

(127 citation statements)

References 105 publications

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“…In eukaryotic cells, protein degradation is mediated by two distincts systems: the nonspecific intravacuolar proteolysis system, which is rather implicated in the stress response (63); and the proteasome ubiquitin-mediated pathway, which has been implicated in specific degradation of abnormal proteins as well as short-lived proteins (18). Ctk2p turnover is not affected in cells deficient in the major vacuolar peptidases (data not shown), whereas it is markedly affected in a mutant strain deficient for two of the proteasome catalytic subunits (pre1 pre2) (48), indicating that Ctk2p destruction is mediated by the proteasome pathway.…”

Section: Discussionmentioning

confidence: 99%

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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The yeast CTDK-I complex has been implicated in phosphorylation of the carboxy-terminal domain of the RNA polymerase II and in transcription control. It is composed of three polypeptides: Ctk1p and Ctk2p, a cyclin-dependent kinase and a C-type cyclin subunit, respectively; and Ctk3p, a third subunit of unknown function. Cyclins are regulatory proteins whose expression is tightly controlled at the protein level. In this study, we examined the regulation of Ctk2p expression in vivo. Surprisingly, unlike what has been described for cell cycle cyclins, steady-state levels of Ctk2p are composed of two relatively abundant forms, one of them phosphorylated. We show that this phosphorylated form is extremely unstable (half-life, 5 min) and that rapid proteolysis of Ctk2p exhibits growth-related regulation. Furthermore, our data establish that similar to the case for other naturally short-lived proteins, Ctk2p degradation is mediated by the ubiquitin-proteasome pathway. This is the first demonstration that a C-type cyclin is phosphorylated and targeted to the proteasome. Strikingly, neither phosphorylation nor destruction of Ctk2p requires its associated kinase Ctk1p, a feature fundamentally different from that which has been observed for cell cycle cyclins.Cyclin-dependent protein kinases (CDKs) were first identified as central regulators of the major transitions of the eukaryotic cell division cycle. Their activity is determined by cyclin binding, by both positive and negative regulatory phosphorylation, and by polypeptide CDK inhibitors (40, 41). A single cyclin-dependent kinase subunit associates sequentially with multiple cyclin partners whose abundance fluctuates during the cell cycle (43). Indeed, one aspect of CDK regulation is triggered by the rapid destruction of their cyclin partners (23). Several studies both in yeast and in mammals have demonstrated that cyclins are selectively degraded by the ubiquitindependent proteasome pathway (9,16,53,67). Substrates are first marked for degradation by conjugation with several molecules of ubiquitin, a highly conserved 76-amino-acid-long polypeptide that is joined reversibly by a covalent linkage.

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Section: Discussionmentioning

confidence: 99%

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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“…6). Vacuoles of Saccharomyces cerevisiae are organelles that have the ability to degrade and recycle unnecessary macromolecular constituents (31,32). For example, addition of glucose to a yeast culture growing on acetate as a carbon source results in rapid inactivation of gluconeogenic enzymes followed by proteolytic degradation of the inactivated enzymes.…”

Section: Discussionmentioning

confidence: 99%

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli

Kuroda

Okuda

Saitoh

et al. 1998

Journal of Biological Chemistry

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Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H ؉ because of respiration and to ATP hydrolysis by the F o F 1 -ATPase. Such current was inhibited by inhibitors of the respiratory chain or F o F 1 -ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.The patch clamp technique is an excellent method to measure ion movement across cell membranes as current (1). An extremely small glass pipette (about 1 m in diameter) is attached to the membranes, and activity of ion translocating proteins (ion channels, ion pumps, or ion transporters) is directly measured. So far, however, this important method has been mainly utilized for studies on animal or plant cells but scarcely for bacterial cells (2). Bacterial cells are usually too small to be measured by this method.Escherichia coli, a Gram-negative bacterium, is the best characterized organism from both biochemical and genetical points of view. Ion pumps and ion transporters in E. coli are biochemically well characterized. Many mutant E. coli cells are available. Thus, genetical manipulations are very easy with this microorganism. Therefore, development of a patch clamp method applicable to E. coli membranes must be extremely valuable. Cells of E. coli are surrounded by an outer membrane and an inner membrane (cytoplasmic membrane) separated by a peptidoglycan layer and a periplasmic space. All of the major ion pumps and ion transporters such as the respiratory chain, F o F 1 -ATPase, various ion transporters, and ion-coupled solute transporters are located in the cytoplasmic membrane. To measure ion translocation via such ion pumps or transporters of the cytoplasmic membrane, we have to overcome the following three hurdles: 1) we have to prepare giant vesicles, the diameter of which must be at least 10 m (this is important to get high success rate and accuracy of measurement), 2) the pipette must be directly accessible to the cytoplasmic membrane, and 3) the substrates or effectors of the ion pumps or transporters must be easily accessible to the active site of the proteins and easily removable from the system.It would be essential to prepare giant protoplasts to overcome the first two hurdles. Many attempts have been made by many research groups to prepare giant bacterial cells or giant protoplasts. So far, however, no giant protoplasts surrounded by cytoplasmic membranes and suitable for patch clamp analysis have been prepared....

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“…This is best illustrated by the use of protease-deficient strains to improve the quality and yields of various heterologous proteins [111]. In general, the secreted recombinant proteins can potentially be proteolytically degraded in the culture medium by extracellular proteases, cell-bound proteases [57] and/or by intracellular proteases from lysed cells.…”

Section: Controlling Proteolysismentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,095

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Review: Biosynthesis and function of yeast vacuolar proteases

Cited by 177 publications

References 105 publications

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli

Heterologous protein production using the Pichia pastoris expression system

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