Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

Mumberg, Dominik; Müller, Rolf; Funk, Martin

doi:10.1016/0378-1119(95)00037-7

Cited by 1,812 publications

(1,520 citation statements)

References 17 publications

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“…The plasmids pXY142-mitodsRED (mitoRED) [32], p412-ADH1 mitoTagBFP (mitoBFP::LEU) [12], p416ADH1 mitotagBFP (mitoBFP::URA), Ruby-Tub1::LEU, and pFA6-link-mKate- spHIS5 [17], pKT127 pFA6-link-yEGFP-KAN, pKT209 pFA6-link-yEGFP-CaURA3, and pKT128 pFA6a–link–yEGFP–SpHIS5 [33], p416-ADH1 and p414-MET25 [34] were described previously. pHIS3p:mRuby2-Tub1 + 3'UTR::HPH (Ruby-Tub1::HYG) (Addgene plasmids #50,633) was a gift from W. Lee, UMass Amherst [35].…”

Section: Methodsmentioning

confidence: 99%

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

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Section: Methodsmentioning

confidence: 99%

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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“…The pre-pro-leader was connected to the α-amylase by fusion PCR of the two segments together using primers LZH015 and LZH039 [53]. The pre-pro-insulin and pre-pro-amylase were cloned into the SpeI/SalI or SpeI/EcoRI sites of p426GPD, respectively, downstream of the constitutive GAPDH promoter [54], to create pYapIns and pYapAmy. Plasmids p426GPD, pYapIns, and pYapAmy were transformed into CEN.PK 113-5D and Δhac1 strains by standard methods [51].…”

Section: Methodsmentioning

confidence: 99%

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

et al. 2012

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BackgroundThe protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).ResultsHAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.ConclusionsApparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

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“…Clone the sequence for yEGFP 15 (less start Methionine) with a C-terminal octa-His tag and a TEV protease site upstream of GFP-8His into a 2 μ vector that harbors a GAL1 promoter and URA selection marker (p424GAL1) 17 . In addition, introduce a SmaI site preceding the TEVp-GFP-8His sequence (Fig.…”

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confidence: 99%

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

et al. 2008

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It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for highresolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 μGFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

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Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

Cited by 1,812 publications

References 17 publications

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

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