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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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Characterization of content mixing between late endosomes and lysosomes. (a) Time course under standard incubation  conditions, i.e., in the presence of cytosol, an ATP-regenerating  system, 1 mM ATP, and 1 mM GTP. Results are expressed at each  time point as the percentage of the total immunoprecipitable  counts that had been formed within membranous compartments  (i.e., which were immunoprecipitable in the presence of biocytin).  (b) Requirements for the content mixing reaction. Results are expressed as percentages of the content mixing obtained on the  same day under the standard conditions, i.e., in the presence of  cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM  GTP. 0.2 mM GMP-PNP or guanosine 5′-O-(3-thiotriphosphate  (GTPγS) replaced GTP where indicated. incbn, incubation.
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Figure 1: Characterization of content mixing between late endosomes and lysosomes. (a) Time course under standard incubation conditions, i.e., in the presence of cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM GTP. Results are expressed at each time point as the percentage of the total immunoprecipitable counts that had been formed within membranous compartments (i.e., which were immunoprecipitable in the presence of biocytin). (b) Requirements for the content mixing reaction. Results are expressed as percentages of the content mixing obtained on the same day under the standard conditions, i.e., in the presence of cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM GTP. 0.2 mM GMP-PNP or guanosine 5′-O-(3-thiotriphosphate (GTPγS) replaced GTP where indicated. incbn, incubation.

Mentions: A cell-free assay to determine endosome–lysosome content mixing was modified from that described previously (Mullock et al., 1994). The modified assay contained rat liver late endosomes preloaded with Av-ASF and rat liver lysosomes preloaded with 125I-bpIgA, together with pig brain cytosol plus an ATP-regenerating system and 1 mM GTP. A time course for content mixing is shown in Fig. 1 a. The assay showed a very reproducible fusion between late endosomes and lysosomes (6.8 ± 0.3% of total immunoprecipitable radiolabeled biotin complexed with avidin in 10 min over 55 experiments). Since lysosomes were freshly prepared for each experiment, all results are presented as a percentage of the standard fusion on the same occasion. Using brain cytosol, which lacks the enzymes to metabolize the glycogen in the lysosomal preparation, the energy dependence of the fusion reaction was much more obvious than that observed previously (Mullock et al., 1994); merely omitting ATP and GTP reduced the fusion by >75% (Fig. 1 b). Content mixing was dependent on the presence of cytosol and required incubation at 37°C (Fig. 1 b).


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)

Characterization of content mixing between late endosomes and lysosomes. (a) Time course under standard incubation  conditions, i.e., in the presence of cytosol, an ATP-regenerating  system, 1 mM ATP, and 1 mM GTP. Results are expressed at each  time point as the percentage of the total immunoprecipitable  counts that had been formed within membranous compartments  (i.e., which were immunoprecipitable in the presence of biocytin).  (b) Requirements for the content mixing reaction. Results are expressed as percentages of the content mixing obtained on the  same day under the standard conditions, i.e., in the presence of  cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM  GTP. 0.2 mM GMP-PNP or guanosine 5′-O-(3-thiotriphosphate  (GTPγS) replaced GTP where indicated. incbn, incubation.
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Related In: Results  -  Collection

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Figure 1: Characterization of content mixing between late endosomes and lysosomes. (a) Time course under standard incubation conditions, i.e., in the presence of cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM GTP. Results are expressed at each time point as the percentage of the total immunoprecipitable counts that had been formed within membranous compartments (i.e., which were immunoprecipitable in the presence of biocytin). (b) Requirements for the content mixing reaction. Results are expressed as percentages of the content mixing obtained on the same day under the standard conditions, i.e., in the presence of cytosol, an ATP-regenerating system, 1 mM ATP, and 1 mM GTP. 0.2 mM GMP-PNP or guanosine 5′-O-(3-thiotriphosphate (GTPγS) replaced GTP where indicated. incbn, incubation.
Mentions: A cell-free assay to determine endosome–lysosome content mixing was modified from that described previously (Mullock et al., 1994). The modified assay contained rat liver late endosomes preloaded with Av-ASF and rat liver lysosomes preloaded with 125I-bpIgA, together with pig brain cytosol plus an ATP-regenerating system and 1 mM GTP. A time course for content mixing is shown in Fig. 1 a. The assay showed a very reproducible fusion between late endosomes and lysosomes (6.8 ± 0.3% of total immunoprecipitable radiolabeled biotin complexed with avidin in 10 min over 55 experiments). Since lysosomes were freshly prepared for each experiment, all results are presented as a percentage of the standard fusion on the same occasion. Using brain cytosol, which lacks the enzymes to metabolize the glycogen in the lysosomal preparation, the energy dependence of the fusion reaction was much more obvious than that observed previously (Mullock et al., 1994); merely omitting ATP and GTP reduced the fusion by >75% (Fig. 1 b). Content mixing was dependent on the presence of cytosol and required incubation at 37°C (Fig. 1 b).

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