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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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ALLN transiently inhibits uptake of 125I-labeled LDL.  Expression of LDL receptors was induced in normal human fibroblasts by standard methods. The cells were preincubated in  the presence (○) or absence (□) of 500 μM ALLN for 1 h before  the addition of 15 μg/ml of 125I-labeled LDL ± 50-fold excess of  cold LDL. The cells were then incubated further for the indicated  time before the amount of internalized [125I]LDL was measured  as described. Each value is the average of duplicate measurements.
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Figure 5: ALLN transiently inhibits uptake of 125I-labeled LDL. Expression of LDL receptors was induced in normal human fibroblasts by standard methods. The cells were preincubated in the presence (○) or absence (□) of 500 μM ALLN for 1 h before the addition of 15 μg/ml of 125I-labeled LDL ± 50-fold excess of cold LDL. The cells were then incubated further for the indicated time before the amount of internalized [125I]LDL was measured as described. Each value is the average of duplicate measurements.

Mentions: If annexin VI is involved in receptor-mediated endocytosis, the results of the in vitro analysis suggest ALLN should inhibit LDL internalization (Fig. 5). Human fibroblasts were incubated for different times in the presence of media containing 125I-labeled LDL, plus or minus the addition of 500 μM of ALLN. Control cells internalized LDL at a constant rate (□) for the first 30–60 min of the incubation, after which the rate began to plateau. The presence of ALLN in the media totally blocked LDL uptake for the first 30 min of incubation (○). Surprisingly, the cells then began to take up LDL, which continued for the duration of the incubation. These results suggested ALLN initially blocks coated pit budding in vivo, but the cells overcome the inhibition by activating an alternative pathway.


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)

ALLN transiently inhibits uptake of 125I-labeled LDL.  Expression of LDL receptors was induced in normal human fibroblasts by standard methods. The cells were preincubated in  the presence (○) or absence (□) of 500 μM ALLN for 1 h before  the addition of 15 μg/ml of 125I-labeled LDL ± 50-fold excess of  cold LDL. The cells were then incubated further for the indicated  time before the amount of internalized [125I]LDL was measured  as described. Each value is the average of duplicate measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132873&req=5

Figure 5: ALLN transiently inhibits uptake of 125I-labeled LDL. Expression of LDL receptors was induced in normal human fibroblasts by standard methods. The cells were preincubated in the presence (○) or absence (□) of 500 μM ALLN for 1 h before the addition of 15 μg/ml of 125I-labeled LDL ± 50-fold excess of cold LDL. The cells were then incubated further for the indicated time before the amount of internalized [125I]LDL was measured as described. Each value is the average of duplicate measurements.
Mentions: If annexin VI is involved in receptor-mediated endocytosis, the results of the in vitro analysis suggest ALLN should inhibit LDL internalization (Fig. 5). Human fibroblasts were incubated for different times in the presence of media containing 125I-labeled LDL, plus or minus the addition of 500 μM of ALLN. Control cells internalized LDL at a constant rate (□) for the first 30–60 min of the incubation, after which the rate began to plateau. The presence of ALLN in the media totally blocked LDL uptake for the first 30 min of incubation (○). Surprisingly, the cells then began to take up LDL, which continued for the duration of the incubation. These results suggested ALLN initially blocks coated pit budding in vivo, but the cells overcome the inhibition by activating an alternative pathway.

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