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Molecular characterization of the protein encoded by the Hermansky-Pudlak syndrome type 1 gene
E C Dell'Angelica1, R C Aguilar, N Wolins
1Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Insights
Hermansky-Pudlak syndrome type 1 involves a cytosolic protein, HPS1p, crucial for lysosome-related organelle function. Its role is distinct from the AP-3 complex pathway.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Hermansky-Pudlak syndrome (HPS) is a group of genetic disorders affecting lysosome-related organelles.
- HPS type 1 results from mutations in a unique gene, with its protein product (HPS1p) previously uncharacterized.
Purpose of the Study:
- Identify and biochemically characterize the HPS1 gene product (HPS1p).
- Determine the subcellular localization and function of HPS1p in relation to lysosome-related organelles.
- Differentiate the HPS1p pathway from the AP-3 adaptor complex pathway in HPS.
Main Methods:
- Biochemical characterization of HPS1p, including electrophoretic mobility and ultracentrifugation.
- Analysis of lysosomal membrane protein trafficking (CD63, Lamp-1) in HPS1p-deficient cells and HPS mouse models.
- Comparison of HPS1p-deficient cells with cells harboring mutations in the AP-3 adaptor complex.
Main Results:
- HPS1p is a ~80 kDa cytosolic protein, peripherally associated with membranes, not an integral membrane protein as predicted.
- HPS1p-deficient cells show normal lysosomal protein trafficking, unlike AP-3 deficient cells which exhibit increased plasma membrane routing.
- Only AP-3 mutant mouse models (pearl, mocha) showed altered Lamp-1 trafficking, distinct from HPS1p-deficient cells.
Conclusions:
- HPS1p is a cytosolic protein involved in lysosome-related organelle biogenesis/function.
- HPS1p acts through a mechanism independent of the AP-3 adaptor complex.
- This study clarifies the distinct molecular pathways underlying different forms of Hermansky-Pudlak syndrome.
Abstract:
Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of approximately 80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was approximately 6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the beta3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1 gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.