Site-specific photo-crosslinking of Hsc70 with the KFERQ pentapeptide motif in a chaperone-mediated autophagy and microautophagy substrate in mammalian cells

Tatsuro Seike; Kazue Terasawa; Takanori Iwata; Jun-Lin Guan; Tetsuro Watabe; Shigeyuki Yokoyama; Miki Hara-Yokoyama

doi:10.1016/j.bbrc.2024.150515

Site-specific photo-crosslinking of Hsc70 with the KFERQ pentapeptide motif in a chaperone-mediated autophagy and microautophagy substrate in mammalian cells

Biochem Biophys Res Commun. 2024 Aug 8:736:150515. doi: 10.1016/j.bbrc.2024.150515. Online ahead of print.

Authors

Tatsuro Seike¹, Kazue Terasawa², Takanori Iwata¹, Jun-Lin Guan³, Tetsuro Watabe⁴, Shigeyuki Yokoyama⁵, Miki Hara-Yokoyama⁶

Affiliations

¹ Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8549, Japan.
² Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8549, Japan; LiberoThera Co., Ltd., 1-9-10 Nihonbashi-horidome-cho, Chuo-ku, Tokyo, 103-0012, Japan.
³ Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.
⁴ Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8549, Japan.
⁵ Department of Structural Biology and Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8549, Japan; Department of Drug Target Protein Research, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano, 390-8621, Japan.
⁶ Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8549, Japan. Electronic address: m.yokoyama.bch@tmd.ac.jp.

PMID: 39128268
DOI: 10.1016/j.bbrc.2024.150515

Abstract

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.

Keywords: Chaperone; Chaperone-mediated autophagy; Hsc70; Microautophagy; Site-specific photo-crosslinking; The KFERQ motif.