Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy

Abstract

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.

Keywords: 70-kilodalton heat shock protein (Hsp70); autophagy; chaperone; endosome; phosphatidylserine.

FIGURE 1.

C terminus of hsc-70 engage phosphatidylserine. a, ultrastructural analysis of the extruded PS, PC, and PC/cholesterol (PC/Chl) liposomes used in the hsc-70 binding assays. b, Western blotting analysis of total hsc-70, as added in the liposome binding assay and the fraction bound to the liposomes. One of three experiments is shown. c, bar graph reporting the average and standard deviation of three experiments as detailed in b. Data were analyzed by one-way ANOVA (***, p < 0.005) and Tukey test. d, Western blotting analysis of liposome-bound hsc-70 in presence and absence of ATP. e, bar graph reporting the average and standard deviation of three experiments as detailed in d. Data were analyzed by one-way ANOVA (***, p < 0.005) and Tukey test. f and g, PS or PC lipid vesicles were immobilized on L1 BIAcore chips, and binding to hsc-70 was monitored. The results confirm that hsc-70 binds to PS but not PC. RU, response units.

FIGURE 2.

NMR studies of the interaction of hsc-70 SBD with DOPS. a, overlay of two-dimensional ¹⁵N-¹H TROSY-HSQC spectra of 45 mm hsc-70(395–604) without (blue) and with (green) 160 mm and 1600 μm DOPS (red). The figure is an enlargement highlighting the DOPS-induced chemical shift perturbation of Lys-601. b, amide group chemical shift perturbations (dNH = √(ΔδN² = ΔδH²) δ) resulting from adding 1.65 mm DOPS to 45 mm hsc-70 SBD in the presence of 165 mm hydrophobic TAU peptide KVQIINKKGCGMGHHHHHH blocking the substrate-binding cleft. c, lysine and arginine residue amide group chemical shift perturbations resulting from adding 1.65 mm DOPS to 45 mm hsc-70 SBD in the presence of 165 mm of the hydrophobic TAU peptide. d, homology model of hsc-70 SBD based on the crystal structure of hsp-70 SBD complexed with NRLLLTG (PDB code 4PO2). The figure is color-coded for the NH chemical shift perturbations resulting from adding 1.65 mm DOPS to 45 mm hsc-70 SBD in the presence of 165 mm hydrophobic TAU peptide. Only shifts for Lys and Arg NH are shown. In green are NH shifts smaller than two S.D.; in orange are 2 S.D. < CSP < 3 S.D., and in red CSP > 3 S.D. Gray indicates unassigned/overlapped. Residues Arg-535, Lys-573, Lys-583, Lys-589, Lys-597, and Lys-601 for which mutagenesis studies were carried out are rendered as sticks. The backbone of the bound NRLLLTG is shown in blue. e, overlay of two-dimensional ¹⁵N-¹H TROSY-HSQC spectra hsc-70(395–604) without (blue, hsc 80 mm) and with sub-equivalent DOPS nano-discs (green, hsc 57 mm, discs 36 mm), and supra-equivalent DOPS nano-discs (red, hsc 44 mm, discs 55 mm). The figure is an enlargement highlighting the DOPS-induced chemical shift perturbation of Lys-569. f, amide group chemical shift perturbations resulting from adding 55 μm DOPS nano-discs to 44 μm hsc-70 SBD in the presence of 50 μm hydrophobic peptide MHHHHHHSSGVDLGTENLYFQ blocking the substrate-binding cleft. g, lysine and arginine residue amide group chemical shift perturbations resulting from adding 55 mm DOPS nano-discs to 44 mm hsc-70 SBD in the presence of 50 mm hydrophobic peptide. h, homology model of hsc-70 SBD. The figure is color-coded for the NH chemical shift perturbations resulting from adding 55 mm DOPS nano-discs to 44 mm hsc-70 SBD in the presence of 50 mm hydrophobic peptide. Only shifts for Lys and Arg NH are shown. In green are NH shifts smaller than 2 S.D.; in orange 2 S.D. < CSP < 3 S.D., in red CSP > 3 S.D. Gray indicates unassigned/overlapped. Residues Arg-535, Lys-573, Lys-583, Lys-589, Lys-597, and Lys-601 for which mutagenesis studies were carried out are rendered as sticks. The backbone of the bound NRLLLTG is shown in blue.

FIGURE 3.

Phosphatidylserine binding of hsc-70 mutants. a, circular dichroism to validate correct folding (secondary and tertiary structures) of hsc-70 mutants (R533A, K535A, K573Q, K583Q, K589Q, K597Q, and K601Q) as compared with wild-type hsc-70. b, Western blotting analysis of total wild-type and mutants hsc-70, as added in the liposome binding assay, and the fraction bound to the liposomes are shown. t, total; b, bound. c, mean and standard deviation of four different liposome-binding experiments, as reported in b. Data were analyzed by one-way ANOVA (*, p < 0.05; ***, p < 0.005) and Tukey test.

FIGURE 4.

Biological role of hsc-70-PS complex in cytosol-to-endosome protein internalization via endosomal MI. a, FACS analysis of gradient-purified late endosomal compartments plus (+) or minus (−) labeling with (5-(octadecanoylamino) fluorescein) lipophilic probe. b, ultrastructural analysis of gradient-purified late endosomes. c, late endosomes, fluorescently labeled as in a, were incubated with recombinant wild-type hsc-70 or hsc-70 mutants proteins. Changes in fluorescence intensities, indicative of hsc-70 binding to the endosome limiting membrane, are reported as mean and standard deviation. Data were analyzed by one-way ANOVA (**, p < 0.01; ***, p < 0.005) and Tukey test. RFU, relative fluorescence units. d, representative immunoblot for GAPDH of endosomes recovered by centrifugation following incubation with GAPDH in the presence of wild type of hsc-70 mutant proteins. Where indicated the protease inhibitors (PI) were added to block GAPDH degradation. Ponceau staining is shown at the bottom to demonstrate equal endosomal content. e and f, quantification of the amount of hsc-70-GAPDH bound (e) and internalized and degraded (f) into endosomes. Internalization and degradation were calculated by discounted GAPDH bound to endosomes in absence of protease inhibitors to that associated with endosomes in the presence of protease inhibitors. Values are expressed as percentage of GAPDH added and are mean and standard deviation (n = 3) of the densitometric analysis of immunoblots as the one shown in d. Data were analyzed by t test (*, p < 0.05). g, representative immunoblot for hsc-70 (top) and GAPDH (bottom) of endosomes incubated with wild type of hsc-70 mutants alone (−) or in the presence of GAPDH (+). h shows one-fifth of the hsc-70 inputs. i, quantification of the amount of hsc-70 bound to endosomes in presence of GAPDH. Values are expressed relative to the amount of hsc-70 bound to endosomes when incubated without GAPDH and are mean and standard deviation (n = 4) of the densitometric analysis of immunoblots as the one shown in g. Data were analyzed by t test (*, p < 0.05).

FIGURE 5.

Homology model of wild-type hsc-70. The model is based on the crystal structures of hsc-70 NBD and hsp-70 (HSPA1) SBD. At right is the NBD; at left is the SBD. The unstructured peptide at the left bottom depicts residues 610–646. a, distribution of positive (blue) and negative (red) residues over hsc-70. The residues Arg-533, Lys-535, Lys-569, Lys-573, Lys-583, Lys-589, Lys-597, and Lys-601 that interact with DOPS and liposomes as defined in this work are shown as spheres. b, surface electrostatic map in the same orientation as a. If a positive/negative charge would touch the red/blue surface, it would perceive an attractive energy of 0.6 kcal/mol. c, known interaction sites or surfaces of hsc-70. ADP is indicated in cyan spheres. The substrate-cleft bound peptide NRLLLTG is shown in red sticks. Blue is the hsc-70 PS binding area defined in this work. Other binding areas are shown as follows. Orange, CHIP-binding site; magenta, the HdJ-binding site in analogy to DnaK-DnaJ; green, binding site of BAG1; yellow, binding site of the MKT077-related hsc-70-modifying compounds. d, as c, but rotated by 90° along the horizontal axis.