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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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Annexin VI binding to attached membranes (B and C)  supports coated pit budding (A). (A) Attached membranes were  incubated for 15 min at 4°C in the presence of either buffer alone  (bars 1–3) or buffer containing 1 nM AnxVIwt (bars 4–6). Membranes were either assayed immediately (bar 4) or warmed to  37°C for 10 min in the presence of either buffer (bars 1 and 5), cytosol alone (bars 2 and 6), or cytosol containing AnxVIwt (bar 3).  Maximum clathrin value was 36,902 cpm/well with a background  of 1,182 cpm/well. (B) Attached membranes were incubated in  the presence of cytosol containing the indicated concentration of  either AnxVIwt (○) or AnxVIΔ192 (□) for 15 min at 4°C. The  membranes were washed and assayed for the amount of bound  annexin VI as described. Background binding of BSA ranged between 3,056 cpm/well and 8,654 cpm/well and was subtracted  from all values. (C) Attached membranes were either not treated  (bar 1) or incubated at 4°C for 15 min in the presence of either  buffer alone (bar 2), buffer plus 1 nM AnxVIwt (bar 3), cytosol  plus 1 nM AnxVIwt (bar 4) or cytosol plus 1 nM AnxVIwt and in 3  nM β-spectrin1–272 (bar 5). Membranes were then assayed for the  amount of annexin VI bound. Background binding to BSA has  been subtracted from all values. All values are the average of  triplicate measurements ± the standard deviation.
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Figure 2: Annexin VI binding to attached membranes (B and C) supports coated pit budding (A). (A) Attached membranes were incubated for 15 min at 4°C in the presence of either buffer alone (bars 1–3) or buffer containing 1 nM AnxVIwt (bars 4–6). Membranes were either assayed immediately (bar 4) or warmed to 37°C for 10 min in the presence of either buffer (bars 1 and 5), cytosol alone (bars 2 and 6), or cytosol containing AnxVIwt (bar 3). Maximum clathrin value was 36,902 cpm/well with a background of 1,182 cpm/well. (B) Attached membranes were incubated in the presence of cytosol containing the indicated concentration of either AnxVIwt (○) or AnxVIΔ192 (□) for 15 min at 4°C. The membranes were washed and assayed for the amount of bound annexin VI as described. Background binding of BSA ranged between 3,056 cpm/well and 8,654 cpm/well and was subtracted from all values. (C) Attached membranes were either not treated (bar 1) or incubated at 4°C for 15 min in the presence of either buffer alone (bar 2), buffer plus 1 nM AnxVIwt (bar 3), cytosol plus 1 nM AnxVIwt (bar 4) or cytosol plus 1 nM AnxVIwt and in 3 nM β-spectrin1–272 (bar 5). Membranes were then assayed for the amount of annexin VI bound. Background binding to BSA has been subtracted from all values. All values are the average of triplicate measurements ± the standard deviation.

Mentions: The site(s) of action for annexin VI may be in the cytosol, at the plasma membrane, or both. To distinguish between these possibilities, we measured the ability of prebound annexin VI to support budding (Fig. 2 A). Membranes were incubated in the presence (Fig. 2 A, bars 4–6) or absence (bars 1–3) of buffer B containing annexin VI for 15 min at 4°C. The membranes were washed and either not warmed (Fig. 2 A, bar 4) or warmed to 37°C in the presence of either inactive cytosol (Fig. 2 A, bars 2 and 6), buffer alone (Fig. 2 A, bars 1 and 5) or inactive cytosol containing AnxVIwt (Fig. 2 A, bar 3). Just as much clathrin loss occurred from membranes preincubated in the presence of AnxVIwt as from untreated membranes that were warmed in cytosol containing AnxVIwt (Fig. 2 A, compare bar 3 with 6). Buffer alone did not support budding under any condition (Fig. 2 A, bars 1 and 5), nor did cytosol in the absence of AnxVIwt (Fig. 2 A, bar 2). These results suggest prebound annexin VI can activate cytosolic factors necessary for the budding reaction.


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)

Annexin VI binding to attached membranes (B and C)  supports coated pit budding (A). (A) Attached membranes were  incubated for 15 min at 4°C in the presence of either buffer alone  (bars 1–3) or buffer containing 1 nM AnxVIwt (bars 4–6). Membranes were either assayed immediately (bar 4) or warmed to  37°C for 10 min in the presence of either buffer (bars 1 and 5), cytosol alone (bars 2 and 6), or cytosol containing AnxVIwt (bar 3).  Maximum clathrin value was 36,902 cpm/well with a background  of 1,182 cpm/well. (B) Attached membranes were incubated in  the presence of cytosol containing the indicated concentration of  either AnxVIwt (○) or AnxVIΔ192 (□) for 15 min at 4°C. The  membranes were washed and assayed for the amount of bound  annexin VI as described. Background binding of BSA ranged between 3,056 cpm/well and 8,654 cpm/well and was subtracted  from all values. (C) Attached membranes were either not treated  (bar 1) or incubated at 4°C for 15 min in the presence of either  buffer alone (bar 2), buffer plus 1 nM AnxVIwt (bar 3), cytosol  plus 1 nM AnxVIwt (bar 4) or cytosol plus 1 nM AnxVIwt and in 3  nM β-spectrin1–272 (bar 5). Membranes were then assayed for the  amount of annexin VI bound. Background binding to BSA has  been subtracted from all values. All values are the average of  triplicate measurements ± the standard deviation.
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Figure 2: Annexin VI binding to attached membranes (B and C) supports coated pit budding (A). (A) Attached membranes were incubated for 15 min at 4°C in the presence of either buffer alone (bars 1–3) or buffer containing 1 nM AnxVIwt (bars 4–6). Membranes were either assayed immediately (bar 4) or warmed to 37°C for 10 min in the presence of either buffer (bars 1 and 5), cytosol alone (bars 2 and 6), or cytosol containing AnxVIwt (bar 3). Maximum clathrin value was 36,902 cpm/well with a background of 1,182 cpm/well. (B) Attached membranes were incubated in the presence of cytosol containing the indicated concentration of either AnxVIwt (○) or AnxVIΔ192 (□) for 15 min at 4°C. The membranes were washed and assayed for the amount of bound annexin VI as described. Background binding of BSA ranged between 3,056 cpm/well and 8,654 cpm/well and was subtracted from all values. (C) Attached membranes were either not treated (bar 1) or incubated at 4°C for 15 min in the presence of either buffer alone (bar 2), buffer plus 1 nM AnxVIwt (bar 3), cytosol plus 1 nM AnxVIwt (bar 4) or cytosol plus 1 nM AnxVIwt and in 3 nM β-spectrin1–272 (bar 5). Membranes were then assayed for the amount of annexin VI bound. Background binding to BSA has been subtracted from all values. All values are the average of triplicate measurements ± the standard deviation.
Mentions: The site(s) of action for annexin VI may be in the cytosol, at the plasma membrane, or both. To distinguish between these possibilities, we measured the ability of prebound annexin VI to support budding (Fig. 2 A). Membranes were incubated in the presence (Fig. 2 A, bars 4–6) or absence (bars 1–3) of buffer B containing annexin VI for 15 min at 4°C. The membranes were washed and either not warmed (Fig. 2 A, bar 4) or warmed to 37°C in the presence of either inactive cytosol (Fig. 2 A, bars 2 and 6), buffer alone (Fig. 2 A, bars 1 and 5) or inactive cytosol containing AnxVIwt (Fig. 2 A, bar 3). Just as much clathrin loss occurred from membranes preincubated in the presence of AnxVIwt as from untreated membranes that were warmed in cytosol containing AnxVIwt (Fig. 2 A, compare bar 3 with 6). Buffer alone did not support budding under any condition (Fig. 2 A, bars 1 and 5), nor did cytosol in the absence of AnxVIwt (Fig. 2 A, bar 2). These results suggest prebound annexin VI can activate cytosolic factors necessary for the budding reaction.

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