Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway

https://www.cell.com/cell/pdf/0092-8674(81)90064-7.pdf

Order of events in the yeast secretory pathway

Publisher Summary This chapter presents the procedure for preparation of high molecular weight RNA. High molecular weight RNA can be isolated from haploid or diploid yeast cells as well as from ascospores. The conditions under which the yeast cells are grown (complete or selective media) do not influence the preparation procedure, although higher yields of RNA are generally obtained from cells grown in rich medium. Procedure requires some special precautions owing to the ubiquitous presence of RNA-degrading enzymes. For this reason it is important to wear gloves during the isolation and preparation procedure, and it is essential to separate all chemicals, tubes, and tips used for RNA preparation from commonly used materials. All glassware has to be baked for 2 hr at 200°; commercially available sterile plastic tubes or tips do not have to be specially pretreated, because they seem to be free of fibonucleases. Because the degradation of RNA by RNases is a time-dependent reaction, it is also advisable to work as fast as possible.

Preparation of high molecular weight RNA.

Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole

https://www.cell.com/cell/pdf/0092-8674(86)90819-6.pdf

Protein sorting in yeast: Mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway

Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a beta-1,3-glucanase with a molecular weight of 55,000. The K(m) for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of beta-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.

Janet H. Scott

Papers

Lyticase: Endoglucanase and Protease Activities That Act Together in Yeast Cell Lysis