Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation

Nolta, Kathleen V.; Rodriguez-Paris, Juan; Steck, Theodore L.

doi:10.1016/0167-4889(94)90196-1

Cited by 38 publications

(45 citation statements)

References 40 publications

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“…Its catalytic activity, like other lysosomal enzymes in D. discoideum [43], was enhanced at pH 6, close to the measured pH of D. discoideum lysosomes [36,38]. Since both phagosomes and pinosomes are acidified by the vacuolar H + -ATPase (Figure 3) [34,37,[44][45][46], the more effective activity of gp70 at lower pH is consistent with our suggested role of gp70 in the hydrolysis of endocytosed nutrients. Possibly the lower pH also plays a role in the transition of gp70 from a crystalline state (Figure 1; esterosomes) to a soluble enzyme when fused with phagosomes and pinosomes.…”

Section: Function Of Gp70 During Vegetative Growthsupporting

confidence: 71%

Role of Esterase gp70 and Its Influence on Growth and Development of Dictyostelium discoideum

Yuan

Chia

2000

Experimental Cell Research

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Gp70 is an esterase originally called crystal protein because of its presence in crystalline structures in aggregation-competent Dictyostelium discoideum cells. Although postulated to break down spore coats, the function of gp70 in vivo was incompletely investigated. Our immunolocalization and biochemical studies of vegetative D. discoideum amoebae show that gp70 was recruited to phagosomes and found in lysosomes. Purified gp70 was effective at hydrolyzing naphthyl substrates with acyl chains typical of lipids and lipopolysaccharides, indicating that the gp70 was involved in digesting endocytosed molecules. The activity of purified gp70 was inhibited by reductants that retarded its electrophoretic mobility and verified the presence of intramolecular disulfide bonds predicted by its amino acid sequence. Compared to wild-type cells, cells overexpressing gp70 were more phagocytically active, had shorter generation times, and produced more fruiting bodies per unit area, while cells lacking gp70 were phagocytically less active with longer doubling times, developed more slowly, and had significantly fewer fruiting bodies per unit area. Consistent with the phenotype of a disrupted metabolism, one-third of the gp70-minus cells were large and multinucleated. Together, these results indicated that despite its crystalline appearance, gp70 was an active esterase involved in both the growth and the development of D. discoideum.

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Section: Function Of Gp70 During Vegetative Growthsupporting

confidence: 71%

Role of Esterase gp70 and Its Influence on Growth and Development of Dictyostelium discoideum

Yuan

Chia

2000

Experimental Cell Research

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“…In mammalian cells, lysosomes represent a terminal endocytic compartment, but in Dictyostelium, lysosomes are a preterminal compartment. The terminal endocytic compartment in Dictyostelium, termed postlysosomes (Aubry et al, 1993;Padh et al, 1993), are relatively novel vesicles that are >2.0 ,tm in diameter and contain a lesser amount of V-H+-ATPase and hydrolases than lysosomes (Rodriguez-Paris et al, 1993;Nolta et al, 1995). Magnetic fractionation of endocytic compartments containing colloidal iron coupled with Western blot analysis indicated Rab7 was highly enriched in lysosomes and postlysosomes, but the protein was not significantly enriched in pinosomes.…”

Section: Discussionmentioning

confidence: 99%

“…The antibodies were then purified with a Rab7 antigen column. Lysosomes were also isolated by a magnetic fractionation procedure from cells pulse labeled for 15 min with iron-coated dextran followed by a 15-min chase (Rodriguez-Paris et al, 1993;Nolta et al, 1995). Membranes of the CV were purified by sucrose density gradient fractionation .…”

Section: Rab7 Localizes To Lysosomes and Postlysosomesmentioning

confidence: 99%

“…A major pathway, after internalization of fluid, involves pinosomes rapidly fusing to form acidic lysosomes, followed by the delivery of fluid phase to larger less acidic postlysosomal vacuoles Aubry et al, 1993;Padh et al, 1993;Nolta et al, 1995). Essentially all of the internalized fluid phase is exocytosed from cells from postlysosomes, beginning approximately 45 min after internalization; no early endosomal recycling compartment has been identified (Aubry et al, 1993;Padh et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

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Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

Buczynski

Bush

Zhang

et al. 1997

MBoC

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The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexpressed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT-and Rab7 Q67L-expressing cells contained a reduced number of vesicles the size of postlysosomes (>2.5 ,um) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, >90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal a-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted a-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H+-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

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“…It remains puzzling why newly geminated KAX3 cells had a much higher frequency of intracellular giant vacuoles (Figures 6 & 7) when compared to that of cultures maintained over 1-2 months. The presence of the H + -ATPase in some of the giant vacuoles ( Figure 5) was consistent with the genesis of these structures from endocytic vesicles, which in D. discoideum are acidified by a vacuolar H + -ATPase (Cardelli et al, 1989;Nolta et al, 1994;Padh et al, 1989Padh et al, , 1991aTemesvari et al, 1996). Our suggestion that giant vacuoles were derived from endosomes agrees with an earlier finding that inhibition of the vacuolar H + -ATPase induces the formation of large intracellular vesicles in D. discoideum (up to 5 μm in diameter) that accumulate FITC-dextran (Temesvari et al, 1996).…”

Section: Discussionmentioning

confidence: 61%

GIANT VACUOLES OBSERVED IN DICTYOSTELIUM DISCOIDEUM

Yuan

Chia

2001

Cell Biology International

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Large intracellular vacuoles, >4μm in diameter and either round or oval‐shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically‐grown strain AX2 (only 1 in 106–108cells). These previously unreported single or multiple ‘giant’ vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT‐Kneg, a strain that lacks an esterase (0.47% of the population). A vacuolar H+‐ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid‐phase marker, fluorescein‐labeled dextran, implying a pinocytotic origin for the vacuoles.

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Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation

Cited by 38 publications

References 40 publications

Role of Esterase gp70 and Its Influence on Growth and Development of Dictyostelium discoideum

Role of Esterase gp70 and Its Influence on Growth and Development of Dictyostelium discoideum

Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

GIANT VACUOLES OBSERVED IN DICTYOSTELIUM DISCOIDEUM

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