Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells.

Makarow, Marja

doi:10.1002/j.1460-2075.1985.tb03861.x

Cited by 58 publications

(37 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…4B), amylase production in the two strains exhibited trends similar to those in the YPD medium (Fig. 3) in that 1D-AAC-VtaM produced more amylase than 1D-AAC until up to around 55 h. It has been reported previously that yeast can take up amylase through endocytosis (51,52). After that point (55 h), the main nutrients supporting cell growth as well as amylase production, the carbon source (glucose) and the added BSA, were depleted, so it was possible that the yeast cells started taking up the produced amylase through endocytosis.…”

Section: ])supporting

confidence: 70%

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Liu

Österlund

et al. 2014

Appl Environ Microbiol

View full text Add to dashboard Cite

cThe increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

show abstract

Section: ])supporting

confidence: 70%

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Liu

Österlund

et al. 2014

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…We showed recently that the yeast S. cerevisiae has a functional endocytic apparatus. In these experiments, VSV, Semliki Forest virus, a-amylase, and FITC-conjugated dextran were used as markers (7,18). Similar findings were reported using Lucifer yellow to demonstrate endocytosis (19).…”

Section: Discussionsupporting

confidence: 56%

Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae.

Makarow¹,

Nevalainen²,

Kääriäinen³

1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The nucleocapsid of vesicular stomatitis virus (VSV) was introduced into the cytoplasm of Saccharomyces cerevisiae by low pH-dependent fusion of the viral envelope with the spheroplast plasma membrane. This led to de novo synthesis of the three major structural proteins of the virusthe G, N, and M proteins-as shown by immunoprecipitation of [35S]methionine-labeled spheroplast lysates. In NaDodSO4/ polyacrylamide gel electrophoresis, M and N proteins comigrated with those of the virion, whereas the yeast-made G protein migrated as two bands with apparent molecular sizes of 60 and 70 kDa. Both polypeptides appeared to be N-glycosylated, since only one polypeptide with the apparent molecular mass of -55 kDa was produced in the presence of tunicamycin. Phase separation into Triton X-114 suggested that the unglycosylated G protein was membrane bound. According to immunofluorescent surface staining of live spheroplasts, at least part of the G protein was transported to the plasma membrane. Spheroplasts expressing the VSV genes could be fused together by low pH to form polykaryons, indicating that G protein synthetized by yeast was fusogenic-i.e., biologically active.Yeast is an attractive host for production of foreign proteins by recombinant DNA technology, since it is able to glycosylate and secrete them. Intracellular transport and posttranslational modifications of soluble enzymes of Saccharomyces cerevisiae have been well-characterized (1), whereas much less is known about membrane proteins. Extensive glycosylation, characteristic of secretory enzymes and mannoproteins of yeast (2, 3), can be suspected to impair biological functions offoreign glycoproteins expressed in this organism. Studies of intracellular transport and glycosylation of heterologous proteins in yeast require laborious recombinant DNA constructions. We have used a different approach to study the direct expression of the proteins of an animal virus in S. cerevisiae, taking advantage of the activation of the particle-associated transcriptase (L protein) of vesicular stomatitis virus (VSV) (4, 5). The nucleocapsid of VSV was introduced into the yeast cytoplasm by fusion of the virus envelope with the spheroplast plasma membrane. The genes encoding the major structural proteins (G, M, and N) were expressed within 30-120 min. The G protein was transported to the plasma membrane where its fusogenic activity could be elicited in acid medium, resulting in polykaryon formation.MATERIALS AND METHODS Cells and Virus Preparations. The strain OL1 (6) of S. cerevisiae was grown in YPD medium (growth medium) containing 1% yeast extract (Oxoid, Basingstoke, U.K.), 2% Bacto-peptone (Difco), and 2% glucose (BDH), at 30°C in a shaker to densities of 4-15 x 107 cells per ml. Spheroplasts were prepared with zymolyase 60,000 (Seikagaku, Tokyo, Japan) as described (7). All media used for spheroplasts contained 1.2 M sorbitol to prevent osmotic lysis. Unlabeled and [35S]methionine-labeled VSV (Indiana serotype) were grown in baby hamster kidney cells and purifi...

show abstract

“…This feature coupled with the significant Ecm29 staining peripheral to the ER/ERGIC compartments prompted us to ask whether Ecm29 also associates with endosomes. HeLa cells were incubated in medium containing Alexa® 488-dextran (39,40), fixed in 2% paraformaldehyde and stained with anti-Ecm29-1 or Ecm29-2 (the epitope for anti-Ecm29-3 is destroyed by paraformaldehyde). The confocal images shown in Fig.…”

Section: Amino Acid Sequence Of Human Ecm29 (Hecm29)-frommentioning

confidence: 99%

Characterization of Mammalian Ecm29, a 26 S Proteasome-associated Protein That Localizes to the Nucleus and Membrane Vesicles

Gorbea

Goellner

Teter

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.

show abstract

Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells.

Cited by 58 publications

References 34 publications

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae.

Characterization of Mammalian Ecm29, a 26 S Proteasome-associated Protein That Localizes to the Nucleus and Membrane Vesicles

Contact Info

Product

Resources

About