scholarly journals A Cell-Free Assay Allows Reconstitution of Vps33p-Dependent Transport to the Yeast Vacuole/Lysosome

1999 ◽  
Vol 146 (1) ◽  
pp. 85-98 ◽  
Author(s):  
Thomas Vida ◽  
Brenda Gerhardt
Keyword(s):  
Yeast Strain ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Free System ◽  
Cell Free System ◽  
Donor And Acceptor ◽  
A Cell ◽  
Pep4 Gene ◽  
Dependent Transport

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as “donor” membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as “acceptor” membranes. When these membranes are incubated together with ATP and cytosolic extracts, ∼50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.

Yeast cell-free system that catalyses joint-molecule formation in a Rad51p- and Rad52p-dependent fashion

2000 ◽  
Vol 347 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Vijayalakshmi NAGARAJ ◽  
David NORRIS
Keyword(s):  
Homologous Recombination ◽  
Specific Activity ◽  
Yeast Cells ◽  
Active Components ◽  
Free System ◽  
Cell Free System ◽  
Molecule Formation ◽  
A Cell

One of the central reactions of homologous recombination is the invasion of a single strand of DNA into a homologous duplex to form a joint molecule. Here we describe the isolation of a cell-free system from meiotic yeast cells that catalyses joint-molecule formation in vitro. The active components in the system required ATP and homologous DNA and operated in both 0.5 and 13 mM MgCl2. When the cell-free system was prepared from rad51/rad51 and rad52/rad52 mutants and joint-molecule formation was assayed at 0.5 mM MgCl2, the specific activity decreased to 6% and 13.8% respectively of the wild-type level. However, when the same mutant extracts were premixed, joint-molecule formation increased 4-8-fold, i.e. the mutant extracts exhibited complementation in vitro. These results demonstrated that Rad51p and Rad52p were required for optimal joint-molecule formation at 0.5 mM MgCl2. Intriguingly, however, Rad51p and Rad52p seemed to be more dispensable at higher concentrations of MgCl2 (13 mM). Further purification of the responsible activity has proven problematical, but it did flow through a sizing column as a single peak (molecular mass 1.2 MDa) that was co-eluted with Rad51p and RFA, the eukaryotic single-stranded DNA-binding protein. All of these characteristics are consistent with the known properties of the reaction in vivo and suggest that the new cell-free system will be suitable for purifying enzymes involved in homologous recombination.

scholarly journals Four journals and a hypothesis: The birth of biochemistry

2007 ◽  
Vol 29 (2) ◽  
pp. 38-40
Author(s):  
Keith Manchester
Keyword(s):  
Clinical Studies ◽  
Yeast Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The separation of the zymase system from yeast cells by Eduard Buchner in 18971, by showing that such a ‘vital’ process as fermentation could be studied in a cell-free system, opened new vistas for biochemistry. The pioneering work of Emil Fischer on carbohydrates, peptides and proteins moreover suggested many new avenues of exploration in the discipline2. An increasing number of investigators into biochemical topics thus began looking for new outlets for publication in journals less dedicated specifically to physiological, chemical or clinical studies – a circumstance which perhaps explains the sudden explosion of new titles in 1905–1906.

scholarly journals Effect of detergents on sterol synthesis in a cell-free system of yeast

1982 ◽  
Vol 23 (6) ◽  
pp. 803-810
Author(s):  
S Hata ◽  
T Nishino ◽  
N Ariga ◽  
H Katsuki
Keyword(s):  
Sterol Synthesis ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of Endosomal Proteolysis in a Cell-free System

1989 ◽  
Vol 264 (10) ◽  
pp. 5392-5399
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
P D Stahl
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals OXIDATION-COUPLED INCORPORATION OF INORGANIC RADIOPHOSPHATE INTO PHOSPHOLIPIDE AND NUCLEIC ACID IN A CELL-FREE SYSTEM

1949 ◽  
Vol 177 (2) ◽  
pp. 775-788 ◽  
Author(s):  
Morris. Friedkin ◽  
Albert L. Lehninger
Keyword(s):  
Nucleic Acid ◽  
Free System ◽  
Cell Free System ◽  
A Cell
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