ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70

Abstract

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Figure 1

AP Release from CCV is dependent on cytosol and ATP. CCV were incubated without (lanes 1 and 3) or with (lane 2 and 4) 65 μg of bovine brain cytosol; and with (lanes 3 and 4) or without (lanes 1 and 2) 800 μM ATP with a regenerating system. Hexokinase and glucose were included in samples without ATP. Released APs, present in the supernatant after centrifugation, were detected on Western blots with polyclonal antibody 0927.

Figure 2

Cytosol titration into AP release assay. (A) CCVs (3.75 μg) were incubated in the presence of an ATP-regenerating system with increasing amounts of cytosol in the absence (open circle) or presence (closed circle) of 0.5 μg of hsc70 or with increasing amounts of hsc70 in the absence of cytosol (closed triangle) for 5 min. at 25°C. AP release from CCV was detected by ELISA after centrifugation at 4°C, as described in MATERIALS AND METHODS. Hsc70 concentration is indicated by the lower abscissa and is equivalent to the amount of hsc70 present in the corresponding amount of cytosol.

Figure 3

Kinetics of AP release at 37°C. CCV were incubated at 37°C for 0–15 min in the presence (solid symbols) or absence (open symbols) of 0.3 μg hsc70 and no additional cytosol (squares), 6.5 μg cytosol (circles), 31 μg cytosol (triangles) as described in Figure 2. Error bars are hidden by the symbol in some cases.

Figure 4

AP release requires Hsc70. (A) CCV were incubated with or without hsc70 as indicated: without additional cytosol (panel 1), with 65 μg of complete cytosol (panel 2), or with 65 μg of hsc70-depleted cytosol (panel 3). AP release (solid bars) was detected by ELISA, and clathrin release (hatched bars) was detected by densitometry of Coomassie- stained SDS-PAGE gels. (B) AP release was measured by ELISA after incubation of CCV with hsc70-depleted cytosol in the absence (open squares) and presence (closed circles) of 0.5 μg of hsc70. Error bars are hidden by the symbol in some cases.

Figure 5

The anti-Hsc70 antibody 3C5 inhibits clathrin but not AP release. CCV were incubated with 65 μg of cytosol, an ATP-regenerating mixture with 800 μM ATP, and 0- to 10-fold molar excess of 3C5 to hsc70. AP release (open triangle) was measured by ELISA, and clathrin release (closed circle) was measured by densitometry of Coomassie-stained SDS-PAGE gels. Error bars are hidden by the symbol in some cases.

Figure 6

Morphological comparison of coated and uncoated CCV. CCV were incubated with ATP (a), 0.5 μg hsc70 (b), 0.5 μg hsc70 and 10 μg cytosol (c), or 10 μg cytosol, 0.5 μg hsc70, and fourfold molar excess of 3C5 (d) under standard conditions. The entire incubation mixtures (i.e., without centrifugation) were applied to Formvar carbon-coated grids and negatively stained with 2% uranyl acetate. Bar, 100 μm .

Figure 7

ATP dependence of AP and clathrin release. CCV were incubated with 65 μg cytosol, hsc70, an ATP-regenerating system, and the indicated amount of ATP. Clathrin release (closed circle, as determined by densitometry of Coomassie-stained gels) and AP release (open triangle, as determined by ELISA) were measured for each sample. Inset shows a double-reciprocal plot for ATP dependence of AP release. Error bars are hidden by the symbol in some cases.

Figure 8

Superdex 200 gel filtration of cytosol. Cytosol (200 μl of 13 mg/ml) was applied to a 24 ml Superdex 200 column and equilibrated and eluted in buffer AK. Fractions (1 ml) were collected. (A) AP release from CCV incubated with hsc70, ATP, and 8 μl of the indicated fraction was detected by ELISA. (B) Protein elution profile. The column was calibrated with β-amylase (200 kDa), BSA (66 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and vitamin B12 (1.4 kDa), which eluted as indicated in panel A.