Arrested maturing multivesicular endosomes observed in a Chinese hamster ovary cell mutant, LEX2, isolated by repeated flow-cytometric cell sorting

Chinese hamster ovary (CHO) cell mutants defective in the disintegration of endocytosed low-density lipoprotein (LDL) were isolated from mutagenized cells by repeated flow-cytometric cell sorting. After seven rounds of cell sorting, we obtained mutant pools, from which nine mutant clones were established. These mutant strains were all recessive, and were categorized into three complementation groups A, B, and C. The previously established CHO mutant, LEX1 (Lysosome-Endosome X1), fell into the complementation group A. One of the newly isolated mutants, LEX2, fell into the complementation group B, and showed slower degradation of RET-LDL than LEX1 cells. LEX2 showed prominence of well-elaborated multivesicular bodies (MVBs), positive for lysosomal glycoprotein-B/cathepsin D and cation-independent mannose 6-phosphate receptor (CI-MPR), yet negative for transferrin receptor or rab7. Endocytosed intact LDL accumulated in these CI-MPR-positive structures starting at 10-15 minutes of internalization and the accumulation reached completion at 20 minutes. Intermixing of separately internalized fluorescent LDLs between the LEX2 MVBs was slow and saturable at a lower level than observed between late endosomes/lysosomes in wild-type or in LEX1 cells. The receptor recycling pathway to the plasma membrane and the acidification of intracellular compartments were normal in LEX2 cells. These results are consistent with the idea that LEX2 cells are defective in the segregation and sequestration of contents at compartments equivalent to the transport intermediates, previously referred to as endosomal carrier vesicles or maturing MVBs. This MVB stage is likely to be an earlier stage than rab7-positive, lysosome-interacting late endosomes observed in LEX1 cells. Thus, LEX1 and LEX2 mutations could be considered as landmarks for these distinct late endocytic stages, and use of these cells in biochemical and molecular genetic analyses would help to understand the as yet unidentified details of late endocytic pathways including the MVB dynamics.