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A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough
Abstract:
Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.
Insights
Dictyostelium discoideum strain HMW-426 exhibits a defect in lysosomal enzyme processing, specifically affecting alpha-mannosidase and beta-glucosidase. This leads to precursors accumulating in the rough endoplasmic reticulum due to conformational changes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Dictyostelium discoideum is a model organism for studying cellular processes.
- Lysosomal enzymes are crucial for cellular waste breakdown.
- Proper protein processing and trafficking are essential for lysosomal function.
Purpose of the Study:
- To investigate the molecular basis of a defect in lysosomal enzyme processing in Dictyostelium discoideum strain HMW-426.
- To determine the cellular localization and processing status of alpha-mannosidase and beta-glucosidase precursors in the mutant strain.
Main Methods:
- Proteolytic processing assays of lysosomal enzyme precursors.
- Endoglycosidase H digestion to assess oligosaccharide modification.
- Subcellular fractionation to determine protein localization.
- Protease susceptibility assays to evaluate protein conformation.
Main Results:
- The HMW-426 mutant is defective in the proteolytic processing of both alpha-mannosidase and beta-glucosidase precursors.
- Oligosaccharide chains on both precursors are sensitive to Endoglycosidase H, indicating a failure to reach the Golgi apparatus.
- Mutant precursors accumulate within the lumen of the rough endoplasmic reticulum.
- The alpha-mannosidase precursor exhibits altered conformation, suggesting a generalized ER export defect.
Conclusions:
- The HMW-426 mutant displays a generalized defect in the transport of lysosomal protein precursors from the rough endoplasmic reticulum.
- Conformational alterations in lysosomal enzyme precursors likely cause this transport defect.
- This study identifies a critical role for proper protein conformation in ER export for lysosomal targeting.