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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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Effects of recombinant wild-type annexin VI (A) and  truncated annexin VI (A and B) on budding of clathrin-coated  pits. (A) Fibroblast membranes were attached at 4°C and then  warmed to 37°C for 10 min in the presence of either buffer alone  or cytosol containing 1 nM of the indicated recombinant protein.  At the end of the incubation, the amount of clathrin loss was  measured as described. Maximum clathrin value was 27,185 cpm/ well of 125I-labeled streptavidin bound with a background of 731  cpm/well. (B) Fibroblast membranes were warmed in cytosol  containing 1 nM wild-type annexin VI (Anx VIwt) plus the indicated concentration of AnxVIΔ192 (□) or BSA (○). Maximum  clathrin value was 32,475 cpm/well with a background of 1,961  cpm/well. All values are the average of triplicate measurements ±  the standard deviation.
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Figure 1: Effects of recombinant wild-type annexin VI (A) and truncated annexin VI (A and B) on budding of clathrin-coated pits. (A) Fibroblast membranes were attached at 4°C and then warmed to 37°C for 10 min in the presence of either buffer alone or cytosol containing 1 nM of the indicated recombinant protein. At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 27,185 cpm/ well of 125I-labeled streptavidin bound with a background of 731 cpm/well. (B) Fibroblast membranes were warmed in cytosol containing 1 nM wild-type annexin VI (Anx VIwt) plus the indicated concentration of AnxVIΔ192 (□) or BSA (○). Maximum clathrin value was 32,475 cpm/well with a background of 1,961 cpm/well. All values are the average of triplicate measurements ± the standard deviation.

Mentions: Coated pit budding from membranes attached to a solid substratum at 4°C is detected using an indirect radioimmune assay that measures the relative amount of clathrin on the membrane (43). Previously, we documented that clathrin loss is an accurate gauge of coated vesicle formation (39). Although annexin VI appears to be essential for budding in vitro, other cytosolic factors are also needed. In an effort to identify these factors, we set up a standard reaction mixture consisting of cytosol deficient in active annexin VI (refer to Materials and Methods for details), various recombinant forms of annexin VI, Ca+2, and ATP (Fig. 1 A). No loss of clathrin was detected when membranes were incubated at 37°C in the presence of buffer (Fig. 1 A, bar 1) and only a modest loss occurred in the presence of inactive cytosol (Fig. 1 A, bar 2). By contrast, when 1 nM recombinant annexin VI (AnxVIwt) was added to the cytosol, membrane clathrin declined by 75% (Fig. 1 A, bar 3). Annexin VI lacking the last four calcium binding repeats (AnxVIΔ379) supported budding to the same extent (Fig. 1 A, bar 4). A recombinant annexin VI missing the last six repeats (AnxVIΔ192), however, lacked budding activity (Fig. 1 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)

Effects of recombinant wild-type annexin VI (A) and  truncated annexin VI (A and B) on budding of clathrin-coated  pits. (A) Fibroblast membranes were attached at 4°C and then  warmed to 37°C for 10 min in the presence of either buffer alone  or cytosol containing 1 nM of the indicated recombinant protein.  At the end of the incubation, the amount of clathrin loss was  measured as described. Maximum clathrin value was 27,185 cpm/ well of 125I-labeled streptavidin bound with a background of 731  cpm/well. (B) Fibroblast membranes were warmed in cytosol  containing 1 nM wild-type annexin VI (Anx VIwt) plus the indicated concentration of AnxVIΔ192 (□) or BSA (○). Maximum  clathrin value was 32,475 cpm/well with a background of 1,961  cpm/well. All values are the average of triplicate measurements ±  the standard deviation.
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Related In: Results  -  Collection

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Figure 1: Effects of recombinant wild-type annexin VI (A) and truncated annexin VI (A and B) on budding of clathrin-coated pits. (A) Fibroblast membranes were attached at 4°C and then warmed to 37°C for 10 min in the presence of either buffer alone or cytosol containing 1 nM of the indicated recombinant protein. At the end of the incubation, the amount of clathrin loss was measured as described. Maximum clathrin value was 27,185 cpm/ well of 125I-labeled streptavidin bound with a background of 731 cpm/well. (B) Fibroblast membranes were warmed in cytosol containing 1 nM wild-type annexin VI (Anx VIwt) plus the indicated concentration of AnxVIΔ192 (□) or BSA (○). Maximum clathrin value was 32,475 cpm/well with a background of 1,961 cpm/well. All values are the average of triplicate measurements ± the standard deviation.
Mentions: Coated pit budding from membranes attached to a solid substratum at 4°C is detected using an indirect radioimmune assay that measures the relative amount of clathrin on the membrane (43). Previously, we documented that clathrin loss is an accurate gauge of coated vesicle formation (39). Although annexin VI appears to be essential for budding in vitro, other cytosolic factors are also needed. In an effort to identify these factors, we set up a standard reaction mixture consisting of cytosol deficient in active annexin VI (refer to Materials and Methods for details), various recombinant forms of annexin VI, Ca+2, and ATP (Fig. 1 A). No loss of clathrin was detected when membranes were incubated at 37°C in the presence of buffer (Fig. 1 A, bar 1) and only a modest loss occurred in the presence of inactive cytosol (Fig. 1 A, bar 2). By contrast, when 1 nM recombinant annexin VI (AnxVIwt) was added to the cytosol, membrane clathrin declined by 75% (Fig. 1 A, bar 3). Annexin VI lacking the last four calcium binding repeats (AnxVIΔ379) supported budding to the same extent (Fig. 1 A, bar 4). A recombinant annexin VI missing the last six repeats (AnxVIΔ192), however, lacked budding activity (Fig. 1 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