Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Abstract

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCI concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCI or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCI or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCI. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. /8-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.

Genetic manipulation of the Brassicaceae smut fungusThecaphora thlaspeos

“…Most protoplasts were obtained using MgSO 4 (Figure 1). Notably, sorbitol and sucrose inhibited cell wall lysis confirming early observations [33,34]. Subsequently, we tested various protoplasting buffers commonly used, in combination with MgSO 4 as the osmotic stabilizer.…”

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

“…Circumstantial evidence lends some support to the location of cryptic ,Bfructofuranosidase in the periplasmic bodies within young cells of S. rouxii (4,5). These vesicular structures are derived from the plasma membrane but remain attached through fine pedicels, thus thwarting separation from protoplasts after cell wall dissolution (4). A pretreatment that could engender independent lysis of periplasmic bodies without concomitant loss of plasma membrane integrity would have experimental utility in the above application, and the possibility of finding a suitable perturbant was an additional impetus for the present investigations.…”

“…Snail enzymes (Sigma Chemical Co., St. Louis, Mo.) were used for cell wall dissolution in the preparation of protoplasts; debris was removed by density gradient centrifugation as previously described (4).…”

“…As discussed in further detail later, the cell wall may provide an effective barrier to large molecules, including the micelles of certain detergents. Accordingly, protoplasts were prepared by established methods (4) and $ubsequently treated with selected detergents. For example, protoplasts in 2.0 M KCl were subject-ed to a range of concentrations of Triton X-100 for 15 min at room temperature.…”

“…Another unusual feature of S. rouxii is the physically cryptic nature of its P-fructofuranosidase (2, 20), an enzyme generally expressed in yeast species by dint of its location in the periplasmic space (1). Circumstantial evidence lends some support to the location of cryptic ,Bfructofuranosidase in the periplasmic bodies within young cells of S. rouxii (4,5). These vesicular structures are derived from the plasma membrane but remain attached through fine pedicels, thus thwarting separation from protoplasts after cell wall dissolution (4).…”