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Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Kamal A, Ying Y, Anderson RG - J. Cell Biol. (1998)

Bottom Line: Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

ABSTRACT
Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

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Endosomes are  mislocalized (A and B) in  cells incubated in the presence of ALLN. Human fibroblasts were incubated in the  presence (B) or absence (A)  of 500 μM ALLN for 1 h at  37°C before the addition of  25 μg/ml of fluorescent LDL.  The cells were incubated an  additional 90 min before direct visualization.
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Figure 7: Endosomes are mislocalized (A and B) in cells incubated in the presence of ALLN. Human fibroblasts were incubated in the presence (B) or absence (A) of 500 μM ALLN for 1 h at 37°C before the addition of 25 μg/ml of fluorescent LDL. The cells were incubated an additional 90 min before direct visualization.

Mentions: We next looked at the fate of the LDL internalized by ALLN-treated cells (Fig 7, A and B). Cells were preincubated in the presence (Fig. 7 B) or absence (Fig. 7 A) of ALLN for 60 min before adding 25 μg/ml of fluorescent LDL to each dish and continuing the incubation an additional 90 min. Untreated cells had numerous large fluorescent vesicles aggregated near the center of the cell (Fig. 7 A). By contrast, the fluorescent vesicles in ALLN-treated cells were smaller, more uniform in size and scattered throughout the cell. We also found that LDL degradation was markedly inhibited in ALLN-treated cells (Table II). Using a standard 5-h uptake assay, ∼50% of the internalized 125I-labeled LDL was degraded in control cells, whereas very little degradation was detected in ALLN-treated cells. Instead, these cells contained twofold more nondegraded 125I-labeled LDL than control cells.


Annexin VI-mediated loss of spectrin during coated pit budding is coupled to delivery of LDL to lysosomes.

Kamal A, Ying Y, Anderson RG - J. Cell Biol. (1998)

Endosomes are  mislocalized (A and B) in  cells incubated in the presence of ALLN. Human fibroblasts were incubated in the  presence (B) or absence (A)  of 500 μM ALLN for 1 h at  37°C before the addition of  25 μg/ml of fluorescent LDL.  The cells were incubated an  additional 90 min before direct visualization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132873&req=5

Figure 7: Endosomes are mislocalized (A and B) in cells incubated in the presence of ALLN. Human fibroblasts were incubated in the presence (B) or absence (A) of 500 μM ALLN for 1 h at 37°C before the addition of 25 μg/ml of fluorescent LDL. The cells were incubated an additional 90 min before direct visualization.
Mentions: We next looked at the fate of the LDL internalized by ALLN-treated cells (Fig 7, A and B). Cells were preincubated in the presence (Fig. 7 B) or absence (Fig. 7 A) of ALLN for 60 min before adding 25 μg/ml of fluorescent LDL to each dish and continuing the incubation an additional 90 min. Untreated cells had numerous large fluorescent vesicles aggregated near the center of the cell (Fig. 7 A). By contrast, the fluorescent vesicles in ALLN-treated cells were smaller, more uniform in size and scattered throughout the cell. We also found that LDL degradation was markedly inhibited in ALLN-treated cells (Table II). Using a standard 5-h uptake assay, ∼50% of the internalized 125I-labeled LDL was degraded in control cells, whereas very little degradation was detected in ALLN-treated cells. Instead, these cells contained twofold more nondegraded 125I-labeled LDL than control cells.

Bottom Line: Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding.The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded.Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

ABSTRACT
Previously we reported that annexin VI is required for the budding of clathrin-coated pits from human fibroblast plasma membranes in vitro. Here we show that annexin VI bound to the NH2-terminal 28-kD portion of membrane spectrin is as effective as cytosolic annexin VI in supporting coated pit budding. Annexin VI-dependent budding is accompanied by the loss of approximately 50% of the spectrin from the membrane and is blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Incubation of fibroblasts in the presence of ALLN initially blocks the uptake of low density lipoprotein (LDL), but the cells recover after 1 h and internalize LDL with normal kinetics. The LDL internalized under these conditions, however, fails to migrate to the center of the cell and is not degraded. ALLN-treated cells have twice as many coated pits and twofold more membrane clathrin, suggesting that new coated pits have assembled. Annexin VI is not required for the budding of these new coated pits and ALLN does not inhibit. Finally, microinjection of a truncated annexin VI that inhibits budding in vitro has the same effect on LDL internalization as ALLN. These findings suggest that fibroblasts are able to make at least two types of coated pits, one of which requires the annexin VI-dependent activation of a cysteine protease to disconnect the clathrin lattice from the spectrin membrane cytoskeleton during the final stages of budding.

Show MeSH
Related in: MedlinePlus