Figure 1. Domain composition and... | Rockefeller University Press

Figure 1.

$Figure 1. Domain composition and reversible polymerization of MANI-FM. (a) Three FM domains followed by an HA tag were fused to the C-terminal end of the Golgi-targeting portion (cytosolic, transmembrane, and luminal stem domains) of mouse MANI. (b) Scheme of MANI-FM polymerization and depolymerization in the absence and presence of AP. (c) To quantify the amount of MANI-FM present in the transfected cells relative to the endogenous protein, transfected HeLa cells were pulsed with radiolabeled amino acids for 24 h, and the endogenous and transfected proteins were immunoprecipitated and examined by autoradiography. Under the immunoprecipitation conditions most of the target proteins were immunoprecipitated. (d) Quantitation of the levels of MANI-FM relative to that of the MANI (c) suggest the levels of MANI-FM varied between two- and threefold that of the endogenous protein. (e) MANI-FM polymerization as revealed using a sedimentation assay. HeLa cells expressing MANI-FM were cultured in the presence of 1 µM AP, and then the AP was washed out for the indicated times before cell lysis and low-speed centrifugation to separate the pellet and supernatant fractions. The MANI-FM in these fractions was then quantified by gel electrophoresis and Western blotting. MANI-FM shifts from the supernatant to the pellet in <15 min. (f) MANI-FM depolymerization. HeLa cells expressing MANI-FM were washed out of AP 15 min (see panel e) and then 1 µM AP was added again to the cells for the indicated times. MANI-FM shifts back to the supernatant in <5 min. Data are mean ± SEM, from three independent experiments.$

Domain composition and reversible polymerization of MANI-FM. (a) Three FM domains followed by an HA tag were fused to the C-terminal end of the Golgi-targeting portion (cytosolic, transmembrane, and luminal stem domains) of mouse MANI. (b) Scheme of MANI-FM polymerization and depolymerization in the absence and presence of AP. (c) To quantify the amount of MANI-FM present in the transfected cells relative to the endogenous protein, transfected HeLa cells were pulsed with radiolabeled amino acids for 24 h, and the endogenous and transfected proteins were immunoprecipitated and examined by autoradiography. Under the immunoprecipitation conditions most of the target proteins were immunoprecipitated. (d) Quantitation of the levels of MANI-FM relative to that of the MANI (c) suggest the levels of MANI-FM varied between two- and threefold that of the endogenous protein. (e) MANI-FM polymerization as revealed using a sedimentation assay. HeLa cells expressing MANI-FM were cultured in the presence of 1 µM AP, and then the AP was washed out for the indicated times before cell lysis and low-speed centrifugation to separate the pellet and supernatant fractions. The MANI-FM in these fractions was then quantified by gel electrophoresis and Western blotting. MANI-FM shifts from the supernatant to the pellet in <15 min. (f) MANI-FM depolymerization. HeLa cells expressing MANI-FM were washed out of AP 15 min (see panel e) and then 1 µM AP was added again to the cells for the indicated times. MANI-FM shifts back to the supernatant in <5 min. Data are mean ± SEM, from three independent experiments.

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