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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 inhibits loss of spectrin (A) and coated pit budding (B) in vitro. (A) Attached membranes were either not  treated (bar 1) or incubated at 37°C for 10 min in the presence of  cytosol (bars 2–5) containing the indicated additions. At the end  of the incubation, the membranes were assayed for the amount of  spectrin as described. (B) Attached membranes were incubated  at 37°C in the presence of either buffer (bar 1) or cytosol (bars 2–5)  containing the indicated additions. At the end of the incubation,  the percent loss of clathrin was measured as described. Maximum  clathrin value was 29,098 cpm/well with a background of 1,114  cpm/well. All values are the average of triplicate measurements ±  the standard deviation.
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Figure 4: ALLN inhibits loss of spectrin (A) and coated pit budding (B) in vitro. (A) Attached membranes were either not treated (bar 1) or incubated at 37°C for 10 min in the presence of cytosol (bars 2–5) containing the indicated additions. At the end of the incubation, the membranes were assayed for the amount of spectrin as described. (B) Attached membranes were incubated at 37°C in the presence of either buffer (bar 1) or cytosol (bars 2–5) containing the indicated additions. At the end of the incubation, the percent loss of clathrin was measured as described. Maximum clathrin value was 29,098 cpm/well with a background of 1,114 cpm/well. All values are the average of triplicate measurements ± the standard deviation.

Mentions: Spectrin-bound annexin VI could regulate the remodeling of membrane spectrin by activating a cytosolic protease. Since calpain I has been implicated in removing spectrin from the membrane (31, 34), we used the cysteine protease inhibitor ALLN to see if it would block annexin VI–dependent removal of spectrin (Fig. 4 A). Compared with untreated membranes (Fig. 4 A, bar 1), annexin VI caused a significant decrease in the amount of spectrin on the membrane (Fig. 4 A, bar 2). This loss of spectrin was markedly inhibited by adding 5 (Fig. 4 A, bar 3) and 10 μM of ALLN (Fig. 4 A, bar 4) to the incubation. The DMSO carrier used to solubilize the ALLN had no effect (Fig. 4 A, bar 5).


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 inhibits loss of spectrin (A) and coated pit budding (B) in vitro. (A) Attached membranes were either not  treated (bar 1) or incubated at 37°C for 10 min in the presence of  cytosol (bars 2–5) containing the indicated additions. At the end  of the incubation, the membranes were assayed for the amount of  spectrin as described. (B) Attached membranes were incubated  at 37°C in the presence of either buffer (bar 1) or cytosol (bars 2–5)  containing the indicated additions. At the end of the incubation,  the percent loss of clathrin was measured as described. Maximum  clathrin value was 29,098 cpm/well with a background of 1,114  cpm/well. All values are the average of triplicate measurements ±  the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132873&req=5

Figure 4: ALLN inhibits loss of spectrin (A) and coated pit budding (B) in vitro. (A) Attached membranes were either not treated (bar 1) or incubated at 37°C for 10 min in the presence of cytosol (bars 2–5) containing the indicated additions. At the end of the incubation, the membranes were assayed for the amount of spectrin as described. (B) Attached membranes were incubated at 37°C in the presence of either buffer (bar 1) or cytosol (bars 2–5) containing the indicated additions. At the end of the incubation, the percent loss of clathrin was measured as described. Maximum clathrin value was 29,098 cpm/well with a background of 1,114 cpm/well. All values are the average of triplicate measurements ± the standard deviation.
Mentions: Spectrin-bound annexin VI could regulate the remodeling of membrane spectrin by activating a cytosolic protease. Since calpain I has been implicated in removing spectrin from the membrane (31, 34), we used the cysteine protease inhibitor ALLN to see if it would block annexin VI–dependent removal of spectrin (Fig. 4 A). Compared with untreated membranes (Fig. 4 A, bar 1), annexin VI caused a significant decrease in the amount of spectrin on the membrane (Fig. 4 A, bar 2). This loss of spectrin was markedly inhibited by adding 5 (Fig. 4 A, bar 3) and 10 μM of ALLN (Fig. 4 A, bar 4) to the incubation. The DMSO carrier used to solubilize the ALLN had no effect (Fig. 4 A, bar 5).

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