Targeting to lysosomes in mammalian cells: the biosynthetic and endocytic pathways

Endogenous or ectopically expressed lysosomal proteins can be detected in their biosynthetic or endocytic pathways by Western blotting of biosynthetic forms in cells, cell fractions, or their culture medium, by pulse-chase radiolabeling accompanied by immunoprecipitation, or by electron or immunofluorescence microscopy. Western blotting and microscopy reveal the steady-state distribution of a protein, whereas pulse-chase studies are required both to identify transient forms and to define the relationship of the biosynthetic forms detected. Targeting to lysosomes can be dramatically affected by synthesis levels and carbohydrate modification, whether the synthesis is upregulated naturally, for example, by cell transformation, or whether it results from ectopic expression. This occurs because a lysosomal protein, unlike a protein expressed in the cytoplasm, must interact with receptors and be packaged into vesicles that mediate its transport though the secretory pathway. Use of microscopy to establish localization is, therefore, a key aspect of characterization of the cellular pathways utilized by lysosomal proteins.