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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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Spectrin loss from membranes (A) accompanies budding (B and C) of coated pits. (A) Attached membranes were  warmed to 37°C for 10 min in the presence of buffer alone (bar  3), buffer plus AnxVIwt (bar 6), cytosol plus AnxVIwt (bar 4), or  cytosol plus AnxVIΔ192 (bar 5). All wells were then assayed for  the presence of spectrin as described. Background was 500 cpm/ well. (B) Attached membranes were incubated for 10 min at 37°C  in the presence of cytosol alone (⋄, dashed line) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of β-spectrin peptide (□). At the end of the incubation, the amount of  clathrin loss was measured as described. Maximum clathrin value  was 30,216 cpm/well with a background of 1,859 cpm/well. (C)  Attached membranes were incubated for 10 min at 37°C in the  presence of cytosol (⋄, on the ordinate) or cytosol containing 1  nM AnxVIwt plus the indicated concentration of rabbit antihuman spectrin serum (□). At the end of the incubation, the amount  of clathrin loss was measured as described. Maximum clathrin  value was 28,125 cpm/well with a background of 1,407 cpm/well.  All values are the average of triplicate measurements ± the standard deviation.
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Figure 3: Spectrin loss from membranes (A) accompanies budding (B and C) of coated pits. (A) Attached membranes were warmed to 37°C for 10 min in the presence of buffer alone (bar 3), buffer plus AnxVIwt (bar 6), cytosol plus AnxVIwt (bar 4), or cytosol plus AnxVIΔ192 (bar 5). All wells were then assayed for the presence of spectrin as described. Background was 500 cpm/ well. (B) Attached membranes were incubated for 10 min at 37°C in the presence of cytosol alone (⋄, dashed line) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of β-spectrin peptide (□). At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 30,216 cpm/well with a background of 1,859 cpm/well. (C) Attached membranes were incubated for 10 min at 37°C in the presence of cytosol (⋄, on the ordinate) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of rabbit antihuman spectrin serum (□). At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 28,125 cpm/well with a background of 1,407 cpm/well. All values are the average of triplicate measurements ± the standard deviation.

Mentions: The binding of annexin VI to spectrin may be required for remodeling of the membrane cytoskeleton before budding of coated pits. A radioimmune assay was used to measure the relative amount of spectrin on the membrane before and after the completion of the budding reaction (Fig. 3 A). Freshly isolated membranes had ∼6 × 103 cpm/well of antispectrin IgG-binding activity (Fig. 3 A, compare bar 1 with 2). Antispectrin IgG binding activity did not change when the membranes were warmed to 37°C in the presence of buffer (Fig. 3 A, bar 3) or buffer plus AnxVIwt (Fig. 3 A, bar 6), indicating no loss of spectrin under these conditions. Binding activity declined by ∼50%, however, when the buffer was replaced with the standard budding mixture containing AnxVIwt (Fig. 3 A, bar 4). Substitution of AnxVIwt with AnxVIΔ192, which does not affect budding (refer to Fig. 1 A), did not stimulate the loss of spectrin (Fig. 3 A, bar 5). Therefore, conditions that support budding of coated pits reduce the level of spectrin that can be detected on the membrane with this radioimmune assay.


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)

Spectrin loss from membranes (A) accompanies budding (B and C) of coated pits. (A) Attached membranes were  warmed to 37°C for 10 min in the presence of buffer alone (bar  3), buffer plus AnxVIwt (bar 6), cytosol plus AnxVIwt (bar 4), or  cytosol plus AnxVIΔ192 (bar 5). All wells were then assayed for  the presence of spectrin as described. Background was 500 cpm/ well. (B) Attached membranes were incubated for 10 min at 37°C  in the presence of cytosol alone (⋄, dashed line) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of β-spectrin peptide (□). At the end of the incubation, the amount of  clathrin loss was measured as described. Maximum clathrin value  was 30,216 cpm/well with a background of 1,859 cpm/well. (C)  Attached membranes were incubated for 10 min at 37°C in the  presence of cytosol (⋄, on the ordinate) or cytosol containing 1  nM AnxVIwt plus the indicated concentration of rabbit antihuman spectrin serum (□). At the end of the incubation, the amount  of clathrin loss was measured as described. Maximum clathrin  value was 28,125 cpm/well with a background of 1,407 cpm/well.  All values are the average of triplicate measurements ± the standard deviation.
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Figure 3: Spectrin loss from membranes (A) accompanies budding (B and C) of coated pits. (A) Attached membranes were warmed to 37°C for 10 min in the presence of buffer alone (bar 3), buffer plus AnxVIwt (bar 6), cytosol plus AnxVIwt (bar 4), or cytosol plus AnxVIΔ192 (bar 5). All wells were then assayed for the presence of spectrin as described. Background was 500 cpm/ well. (B) Attached membranes were incubated for 10 min at 37°C in the presence of cytosol alone (⋄, dashed line) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of β-spectrin peptide (□). At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 30,216 cpm/well with a background of 1,859 cpm/well. (C) Attached membranes were incubated for 10 min at 37°C in the presence of cytosol (⋄, on the ordinate) or cytosol containing 1 nM AnxVIwt plus the indicated concentration of rabbit antihuman spectrin serum (□). At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 28,125 cpm/well with a background of 1,407 cpm/well. All values are the average of triplicate measurements ± the standard deviation.
Mentions: The binding of annexin VI to spectrin may be required for remodeling of the membrane cytoskeleton before budding of coated pits. A radioimmune assay was used to measure the relative amount of spectrin on the membrane before and after the completion of the budding reaction (Fig. 3 A). Freshly isolated membranes had ∼6 × 103 cpm/well of antispectrin IgG-binding activity (Fig. 3 A, compare bar 1 with 2). Antispectrin IgG binding activity did not change when the membranes were warmed to 37°C in the presence of buffer (Fig. 3 A, bar 3) or buffer plus AnxVIwt (Fig. 3 A, bar 6), indicating no loss of spectrin under these conditions. Binding activity declined by ∼50%, however, when the buffer was replaced with the standard budding mixture containing AnxVIwt (Fig. 3 A, bar 4). Substitution of AnxVIwt with AnxVIΔ192, which does not affect budding (refer to Fig. 1 A), did not stimulate the loss of spectrin (Fig. 3 A, bar 5). Therefore, conditions that support budding of coated pits reduce the level of spectrin that can be detected on the membrane with this radioimmune assay.

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