Molecular forms of yeast invertase

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Yeast invertase is a key enzyme involved in sucrose hydrolysis.
• Understanding the molecular forms and secretion pathway of yeast invertase is crucial for cellular processes.

Purpose of the Study:

• To investigate the molecular forms of yeast invertase and their distribution.
• To elucidate the role of glycosylation in yeast invertase secretion.
• To study the effects of 2-deoxy-D-glucose and cycloheximide on invertase biosynthesis and distribution.

Main Methods:

• Gel filtration on Sephadex G-200 to separate molecular forms.
• Ion-exchange chromatography and isoelectric focusing for invertase separation.
• Protoplasting to analyze intracellular and extracellular enzyme forms.
• Treatment with 2-deoxy-D-glucose and cycloheximide to study biosynthesis and glycosylation.

Main Results:

• Demonstrated a spectrum of yeast invertase molecular forms, from light (carbohydrate-free) to heavy (50% carbohydrate).
• Observed that intermediate forms represent sequential mannose addition during secretion.
• 2-deoxy-D-glucose treatment inhibited glycosylation and secretion, leading to accumulation of light and intermediate forms intracellularly.
• Cycloheximide, while blocking synthesis, allowed glycosylation to proceed, accumulating heavy forms from light ones.

Conclusions:

• Yeast invertase secretion involves sequential glycosylation, forming a spectrum of molecular weights.
• Glycosylation is essential for the proper secretion of heavy invertase from yeast cells.
• 2-deoxy-D-glucose effectively inhibits yeast invertase glycosylation and secretion, causing intracellular accumulation of precursor forms.