The Disordered C-Terminus of the Chaperone DnaK Increases the Competitive Fitness of Pseudomonas putida and Facilitates the Toxicity of GraT

Chaperone proteins are crucial for proper protein folding and quality control, especially when cells encounter stress caused by non-optimal temperatures. DnaK is one of such essential chaperones in bacteria. Although DnaK has been well characterized, the function of its intrinsically disordered C-terminus has remained enigmatic as the deletion of this region has been shown to either enhance or reduce its protein folding ability. We have shown previously that DnaK interacts with toxin GraT of the GraTA toxin-antitoxin system in Pseudomonas putida. Interestingly, the C-terminal truncation of DnaK was shown to alleviate GraT-caused growth defects. Here, we aim to clarify the importance of DnaK in GraT activity. We show that DnaK increases GraT toxicity, and particularly important is the negatively charged motif in the DnaK C-terminus. Given that GraT has an intrinsically disordered N-terminus, the assistance of DnaK is probably needed for re-modelling the toxin structure. We also demonstrate that the DnaK C-terminal negatively charged motif contributes to the competitive fitness of P. putida at both high and optimal growth temperatures. Thus, our data suggest that the disordered C-terminal end of DnaK enhances the chaperone functionality.

Download Full-text

Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0463 ◽

2011 ◽

Vol 22 (24) ◽

pp. 4726-4739 ◽

Cited By ~ 37

Author(s):

Noa Furth ◽

Or Gertman ◽

Ayala Shiber ◽

Omri S. Alfassy ◽

Itamar Cohen ◽

...

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Protein Folding ◽

Nuclear Envelope ◽

Reverse Genetics ◽

Point Mutations ◽

Hydrophobic Surface ◽

Structural Features ◽

Er Quality Control ◽

C Terminus

Proper functioning of the protein-folding quality control network depends on the network's ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell homeostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control–associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone–dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.

Download Full-text

Intrinsic disorder codes for leaps of protein expression

10.1101/2020.12.08.407247 ◽

2020 ◽

Author(s):

Chi-Ning Chuang ◽

Tai-Ting Woo ◽

Shih-Ying Tsai ◽

Wan-Chen Li ◽

Chia-Ling Chen ◽

...

Keyword(s):

Quality Control ◽

Protein Folding ◽

Protein Expression ◽

Posttranslational Modifications ◽

Protein Quality ◽

Intrinsic Disorder ◽

Protein Homeostasis ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Regulate Protein

AbstractIntrinsically disordered regions (IDRs) are protein sequences lacking fixed or ordered three-dimensional structures. Many IDRs are endowed with important molecular functions such as physical interactions, posttranslational modifications or solubility enhancement. We reveal that several biologically important IDRs can act as N-terminal fusion carriers to promote target protein folding or protein quality control, thereby enhancing protein expression. This nanny function has a reasonably strong correlation with high S/T/Q/N amino acid content in IDRs and it is tunable (e.g., via phosphorylation) to regulate protein homeostasis. We propose a hypothesis that “N-terminal intrinsic disorder facilitates abundance” (NIDFA) to explain how some yeast proteins use their N-terminal IDRs (N-IDRs) to generate high levels of protein product. These N-IDRs are versatile toolkits for functional divergence in signaling and evolution.SignificanceDisorder within an otherwise well-structured protein is mostly found in intrinsically disordered regions (IDRs). IDRs can provide many advantages to proteins, including: (1) mediating protein-protein or protein-peptide interactions by adopting different conformations; (2) facilitating protein regulation via diverse posttranslational modifications; and (3) regulating the half-lives of proteins that have been targeted for proteasomal degradation. Here, we report that several biologically important IDRs in S. cerevisiae can act as N-terminal fusion carriers to promote target protein folding or protein quality control, thereby enhancing protein expression. We demonstrate by genetic and bioinformatic analyses that this nanny function is well correlated with high content of serine, threonine, glutamine and asparagine in IDRs and is tunable (e.g., via phosphorylation) to regulate protein homeostasis.

Download Full-text

Phosphorylation of Serine 114 on Atg32 mediates mitophagy

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0145 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3206-3217 ◽

Cited By ~ 127

Author(s):

Yoshimasa Aoki ◽

Tomotake Kanki ◽

Yuko Hirota ◽

Yusuke Kurihara ◽

Tetsu Saigusa ◽

...

Keyword(s):

Signal Transduction ◽

Quality Control ◽

Significant Role ◽

Coiled Coil ◽

Adaptor Protein ◽

Mitochondrial Quality Control ◽

Signal Transduction Cascade ◽

Coiled Coil Domain ◽

C Terminus ◽

N Terminus

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11–Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11–Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100–120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11–Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.

Download Full-text

The human disease gene CLEC16A encodes an intrinsically disordered protein region required for mitochondrial quality control

10.1101/2021.09.03.458272 ◽

2021 ◽

Author(s):

Morgan A. Gingerich ◽

Xueying Liu ◽

Biaoxin Chai ◽

Gemma L. Pearson ◽

Michael P. Vincent ◽

...

Keyword(s):

Quality Control ◽

Human Disease ◽

Intrinsically Disordered Protein ◽

Mitochondrial Quality Control ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Functional Regions ◽

C Terminus ◽

Intrinsically Disordered Protein Region

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic β-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.

Download Full-text

The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity

Biochemical Journal ◽

10.1042/bj20150931 ◽

2016 ◽

Vol 473 (2) ◽

pp. 135-144 ◽

Cited By ~ 39

Author(s):

Glenn R. Masson ◽

Olga Perisic ◽

John E. Burke ◽

Roger L. Williams

Keyword(s):

Substrate Specificity ◽

Tumour Suppressor ◽

Membrane Activity ◽

Intrinsically Disordered ◽

C Terminus ◽

N Terminus ◽

Unstructured Regions

The unstructured regions found at the C-terminus of the tumour suppressor PTEN and the N-terminus PTEN-L can switch the enzymes' substrate specificity from soluble to membrane-embedded, and can also dramatically alter the enzymes' affinity for membranes.

Download Full-text