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Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Mullock BM, Bright NA, Fearon CW, Gray SR, Luzio JP - J. Cell Biol. (1998)

Bottom Line: Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein.We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes.The consequences of this fusion to the maintenance and function of lysosomes are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom.

ABSTRACT
Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

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Quantitation of cathepsin L and avidin immunolabeled  organelles of ultrathin frozen sections from the fractions illustrated in Fig. 4. Results are presented as a mean ± the range  (lysosome fraction) or SEM (hybrid and endosome fractions).  Cathepsin L+/avidin+ structures were significantly enriched in  the hybrid fraction compared to the lysosome and endosome  fractions, * P < 0.01. The cathepsin L+/avidin− structures in the  lysosome fraction were electron dense, whereas similarly labeled  structures in the endosome fraction were electron lucent, suggesting that the latter may be vesicles and organelles from the route  of delivery of newly synthesized cathepsin to lysosomes.
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Figure 3: Quantitation of cathepsin L and avidin immunolabeled organelles of ultrathin frozen sections from the fractions illustrated in Fig. 4. Results are presented as a mean ± the range (lysosome fraction) or SEM (hybrid and endosome fractions). Cathepsin L+/avidin+ structures were significantly enriched in the hybrid fraction compared to the lysosome and endosome fractions, * P < 0.01. The cathepsin L+/avidin− structures in the lysosome fraction were electron dense, whereas similarly labeled structures in the endosome fraction were electron lucent, suggesting that the latter may be vesicles and organelles from the route of delivery of newly synthesized cathepsin to lysosomes.

Mentions: ImmunoEM was carried out on membrane-bound organelles isolated from the lysosomal position (45%/20% Nycodenz interface) and hybrid organelle position (Nycodenz/Ficoll interface) of the step gradient after centrifugation of endosome–lysosome content mixing assay material. When the content mixing assay had been incubated for 10 min at 37°C, the organelles recovered from the Nycodenz/ Ficoll interface were enriched in structures containing both endocytosed avidin and lysosome-derived cathepsin L compared to structures containing both labels in either endosome or lysosome fractions (Fig. 3). There was insufficient bpIgA in these structures to be detected by immunoEM with available antibodies.


Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent.

Mullock BM, Bright NA, Fearon CW, Gray SR, Luzio JP - J. Cell Biol. (1998)

Quantitation of cathepsin L and avidin immunolabeled  organelles of ultrathin frozen sections from the fractions illustrated in Fig. 4. Results are presented as a mean ± the range  (lysosome fraction) or SEM (hybrid and endosome fractions).  Cathepsin L+/avidin+ structures were significantly enriched in  the hybrid fraction compared to the lysosome and endosome  fractions, * P < 0.01. The cathepsin L+/avidin− structures in the  lysosome fraction were electron dense, whereas similarly labeled  structures in the endosome fraction were electron lucent, suggesting that the latter may be vesicles and organelles from the route  of delivery of newly synthesized cathepsin to lysosomes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140175&req=5

Figure 3: Quantitation of cathepsin L and avidin immunolabeled organelles of ultrathin frozen sections from the fractions illustrated in Fig. 4. Results are presented as a mean ± the range (lysosome fraction) or SEM (hybrid and endosome fractions). Cathepsin L+/avidin+ structures were significantly enriched in the hybrid fraction compared to the lysosome and endosome fractions, * P < 0.01. The cathepsin L+/avidin− structures in the lysosome fraction were electron dense, whereas similarly labeled structures in the endosome fraction were electron lucent, suggesting that the latter may be vesicles and organelles from the route of delivery of newly synthesized cathepsin to lysosomes.
Mentions: ImmunoEM was carried out on membrane-bound organelles isolated from the lysosomal position (45%/20% Nycodenz interface) and hybrid organelle position (Nycodenz/Ficoll interface) of the step gradient after centrifugation of endosome–lysosome content mixing assay material. When the content mixing assay had been incubated for 10 min at 37°C, the organelles recovered from the Nycodenz/ Ficoll interface were enriched in structures containing both endocytosed avidin and lysosome-derived cathepsin L compared to structures containing both labels in either endosome or lysosome fractions (Fig. 3). There was insufficient bpIgA in these structures to be detected by immunoEM with available antibodies.

Bottom Line: Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein.We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes.The consequences of this fusion to the maintenance and function of lysosomes are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom.

ABSTRACT
Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37 degrees C, late endosome-lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide-sensitive factor-dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

Show MeSH
Related in: MedlinePlus