Stepwise Activity of ClpY (HslU) Mutants in the Processive Degradation of Escherichia coli ClpYQ (HslUV) Protease Substrates

Abstract

In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.

Escherichia coli Proteome Microarrays Identified the Substrates of ClpYQ Protease

“…The E. coli K12 Proteome Chip Assays-To reduce nonspecific binding, the chip was blocked with 1% bovine serum albumin. 0.5 M ClpY (27) or ClpY (⌬IϩGly) (22,27) in TBS-T (Tris-buffered saline contains 0.05% Tween 20) contains 5 mM ATP and 1% bovine serum albumin were used to probe the chips in a hybridization chamber with shaking at room temperature for 1 h. After incubation, the chips were washed with TBS-T, and incubated with anti-ClpY antibody (22) for another 1 h. After washed with TBS-T again, DyLight 649 (Thermo Scientific, Waltham, MA) labeled goat anti-rabbit antibodies (Abcam, Cambridge, England) were used to probe the chips at room temperature for 30 min. Finally, the chips were washed several times with TBS-T and distilled water.…”

“…The restrictive enzymes EcoRI and HindIII were used to cleavage the gene from pB42AD-ybaB plasmid, and the gene was then ligated to pMal-c2x (NEB®) and pTH18kr-mbp vectors (35), which were prepared using the same restrictive enzymes, to construct pMal-c2x-ybaB, and pTH18kr-mbp-ybaB. pET21a-(6x his tag)-clpY and pET21a-clpQ-(6x his-tag), the expression vectors used for protein purification of His(6x)-ClpY and ClpQ-His(6x) were constructed by Hsieh et, al (27). The vectors were transformed into E. coli and then screened using a growth medium containing ampicillin (pTH18krmbp was screened by kanamycin).…”

“…Measurement of K d Between MBP-YbaB and ClpY-A QCM (Affinix QN ) was employed to measure the K d values of the YbaB-ClpY protein interactions, which were expressed in the transformed E. coli cells through pMal-ybaB and pET21a (6x his tag)-clpY (27). The MBP-YbaB and His-ClpY were purified using amylose resin (New England Biolabs, NEB, Ipswich, MA) and Nickel resin (Talon resin, Takara, Kusatsu, Shiga, Japan), respectively.…”

“…The C domain involves aggregating the atoms in the ClpY and ClpQ complexes (22)(23)(24). Previous studies on how ClpY recognizes substrates have revealed that the G 90 Y 91 V 92 G 93 pore motif (pore 1 site) located at the center pore of ClpY is conservative and essential for unfolding and transporting substrates (25)(26)(27).…”

“…Currently, few substrates have been identified for the ClpYQ protease in comparison with other ATP-dependent proteases (25)(26)(27). Because the substrate of a protease can reflect its role in a bacterial cell, identifying potential enzyme substrates is essential in protease research.…”