Abstract
A new role is reported for CP12, a highly unfolded and flexible protein, mainly known for its redox function with A(4) glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized CP12 can prevent the in vitro thermal inactivation and aggregation of GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not redox-dependent. The protection is specific to CP12, because other proteins, such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific chaperone, since it does not protect other proteins, such as catalase, alcohol dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is necessary to prevent the aggregation and inactivation, since the mutant C66S that does not form any complex with GAPDH cannot accomplish this protection. Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is partially able to protect and to slow down the inactivation and aggregation. Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox function but also behaves as a specific "chaperone-like protein" for GAPDH, although a stable and not transitory interaction is observed. This new function of CP12 may explain why it is also present in complexes involving A(2)B(2) GAPDHs that possess a regulatory C-terminal extension (GapB subunit) and therefore do not require CP12 to be redox-regulated.
Highlights
CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria [1, 2], higher plants [3], the diatom Asterionella formosa [4, 5], and green [1] and red algae [6]
The A4 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) lack these regulatory cysteine residues [13, 14, 17,18,19,20], they are redox-regulated through its interaction with CP12, since the C terminus of this small protein resembles the C-terminal extension of the GapB subunit
We studied the interactions of GAPDH with immobilized reduced or oxidized CP12 using HBS-EP running buffer (BiaCore), 0.1 mM NAD, pH 7.5, at 20 l/min using a BiaCore 2000 apparatus (Uppsala, Sweden)
Summary
CP12 is a small 8.2-kDa protein present in the chloroplasts of most photosynthetic organisms, including cyanobacteria [1, 2], higher plants [3], the diatom Asterionella formosa [4, 5], and green [1] and red algae [6]. There are many functional categories of IUPs [22, 33] They can be, for instance, involved in permanent binding and have (i) a scavenger role, neutralizing or storing small ligands; (ii) an assembler role by forming complexes; and (iii) an effector role by modulating the activity of a partner molecule [33]. These functions are not exclusive; CP12 can form a stable complex with GAPDH, regulating its redox properties [8, 34, 35], and can bind a metal ion [36, 37]. CP12, a Permanent Chaperone-like Protein for GAPDH if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate [41,42,43,44] and a substrate commonly used in chaperone studies [45, 46]
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