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Two distinct subfractions in isolated Saccharomyces cerevisiae plasma membranes
This study describes a method to isolate two distinct plasma membrane fractions from yeast cells. Researchers used a series of biochemical treatments including cell wall digestion, lectin binding, and density gradient centrifugation. They found that both membrane fractions retained key functional properties like ATPase activity and surface labeling. The purified membranes showed similar enzymatic behavior despite coming from different density gradients. Electrophoretic analysis revealed seven major proteins on the plasma membrane surface. These findings suggest that yeast plasma membranes have a complex, heterogeneous structure.
Area of Science:
- Cell membrane biochemistry
- Fungal cell biology
- Membrane fractionation techniques
Background:
Understanding plasma membrane composition remains a challenge in cell biology. Prior research has shown that yeast cells have complex membrane structures, but isolating pure membrane fractions has proven difficult. Techniques like lyticase digestion and density gradient centrifugation have been used before, but purity levels remained low. This gap motivated researchers to refine purification methods. No prior work had resolved distinct subfractions within the same membrane preparation. Earlier studies suggested membrane heterogeneity, but lacked clear separation. Researchers needed a way to distinguish between membrane populations. This paper contributes by using sequential biochemical treatments.
Purpose Of The Study:
This study aimed to isolate and characterize distinct plasma membrane fractions in yeast. Researchers sought to improve purification methods beyond previous approaches. The specific problem addressed was membrane heterogeneity in Saccharomyces cerevisiae. A secretion-blocked mutant was used to control protein export. The goal was to separate membrane populations using density gradients. Researchers wanted to measure ATPase activity in different fractions. They also aimed to identify surface-labeled proteins via electrophoresis. This approach allowed comparison between wild-type and mutant membranes.
Main Methods:
Researchers began by digesting cell walls with lyticase. Spheroplasts were coated with concanavalin A for stabilization. After lysis, alpha-methylmannoside was added to remove lectin-bound material. Sonication at high salt concentration disrupted membranes. A Renografin density gradient separated membrane fractions. Two distinct bands sedimented at 1.15 and 1.17 g/cm3. ATPase activity was measured using vanadate-sensitive assays. Two-dimensional gel electrophoresis analyzed iodinated proteins.
Main Results:
Two purified membrane fractions were obtained at 1.15 and 1.17 g/cm3 densities. ATPase activity recovery ranged from 11 to 18% of total. Surface labeling showed 17 to 29% recovery in membrane fractions. Both fractions retained 125I surface label and ATPase function. ATPase activity had a pH optimum of 5.5 in both samples. The apparent Vmax was 360 to 560 nmol/min/mg protein. ATPase Km for ATP was measured at 0.7 mM. Two-dimensional gels revealed seven major plasma membrane proteins.
Conclusions:
The study demonstrated two distinct plasma membrane subfractions in yeast. Both fractions retained key enzymatic and labeling properties. ATPase activity levels were comparable between wild-type and mutant. Seven major proteins were identified via electrophoretic analysis. The purification method achieved higher yields than previous approaches. Membrane heterogeneity was confirmed through density gradient separation. Surface labeling confirmed membrane integrity in both fractions. These findings suggest complex membrane organization in yeast cells.
Frequently Asked Questions
Researchers used lyticase digestion, concanavalin A coating, and Renografin density gradient centrifugation to separate two distinct membrane fractions.
Vanadate-sensitive ATPase activity was quantified, showing yields of 11 to 18% recovery in the purified membrane fractions.
Alpha-methylmannoside was used to remove concanavalin A-bound material after lysis, ensuring cleaner membrane fractions.
The analysis identified seven major proteins on the plasma membrane surface through 125I labeling.
Both membrane fractions showed ATPase activity with a pH optimum of 5.5.
The ATPase specific activity was not decreased in the mutant plasma membrane compared to wild-type samples.