The Ccl1-Kin28 kinase complex regulates autophagy under nitrogen starvation.

Starvation triggers global alterations in the synthesis and turnover of proteins. Under such conditions, the recycling of essential nutrients by using autophagy is indispensable for survival. By screening known kinases in the yeast genome, we newly identified a regulator of autophagy, the Ccl1-Kin28 kinase complex (the equivalent of the mammalian cyclin-H-Cdk7 complex), which is known to play key roles in RNA-polymerase-II-mediated transcription. We show that inactivation of Ccl1 caused complete block of autophagy. Interestingly, Ccl1 itself was subject to proteasomal degradation, limiting the level of autophagy during prolonged starvation. We present further evidence that the Ccl1-Kin28 complex regulates the expression of Atg29 and Atg31, which is crucial in the assembly of the Atg1 kinase complex. The identification of this previously unknown regulatory pathway sheds new light on the complex signaling network that governs autophagy activity.

The Zα domain of fish PKZ converts DNA hairpin with d(GC)(n) inserts to Z-conformation.

PKZ, protein kinase containing Z-DNA domains, is a novel member of the vertebrate eIF2α kinase family. Containing a catalytic domain in C-terminus and two Z-DNA binding domains (Zα1 and Zα2) in N-terminus, PKZ can be activated through the binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα remains to be established. Here, to understand the impact of PKZ Zα on DNA conformational transition, wild-type Zα1Zα2 and 11 mutant proteins were expressed and purified. At the same time, several different lengths of DNA hairpins-d(GC)nT4(GC)n (n = 2-6) and an RNA hairpin-r(GC)6T4(GC)6 were synthesized. The effects of Zα1Zα2 and mutant proteins on the conformation of these synthetic DNA or RNA hairpins were investigated by using circular dichroism spectrum and gel mobility shift assays. The results showed that DNA hairpins retained a conventional B-DNA conformation in the absence of Zα1Zα2, while some of the DNA hairpins (n≥3) were converted to Z-conformation under Zα1Zα2 induction. The tendency was proportionally associated with the increasing amount of GC repeat. In comparison with Zα1Zα2, Zα1Zα1 rather than Zα2Zα2 displayed a higher ability in converting d(GC)6T4(GC)6 from B- to Z-DNA. These results demonstrated that Zα1 sub-domain played a more essential role in the process of B-Z conformational transition than Zα2 sub-domain did. Mutant proteins (K34A, N38A, R39A, Y42A, P57A, P58A, and W60A) could not convert d(GC)6T4(GC)6 into Z-DNA, whereas S35A or K56A retained some partial activities. Interestingly, Zα1Zα2 was also able to induce r(GC)6T4(GC)6 RNA from A-conformation to Z-conformation under appropriate conditions.

The Zα domain of fish PKZ facilitates the B-Z conformational transition of oligonucleotide DNAs with d(GC)(n) inserts.

PKZ (PKR-like) was discovered as a member of elF2α kinase family in fish, which possesses a conserved catalytic domain of an eIF2α kinase in C-terminal and also two Z-DNA-binding domains (Zα1 and Zα2) in N-terminal. PKZ can be activated through binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα still remains unclear. To investigate a molecular mechanism of how PKZ Zα interacts with Z-DNA, we expressed Zα polypeptide Zα1α2 in Escherichia coli Rosetta strain and purified by affinity chromatography on Ni-NTA resin. Different lengths of oligonucleotide DNAs with various inserts, namely d(GC)(n) (n = 6, 8, 10, 13), d(TA)(n) (n = 6, 10), non-d(GC), and non-d(TA), were designed and synthesized. Circular dichroism spectrum and gel mobility shift assays were used to investigate the effects of Zα1α2 on the conformational transition of different oligonucleotide DNAs. Results showed that oligonucleotide DNAs retained a conventional B-DNA conformation in the absence of Zα1α2. With the increasing amount of Zα1α2 titration, d(GC)(n) were recognized and converted to Z-DNA conformation to some degree. With increasing the repeat number (from n = 6 to n = 13), the tendency of conformational transition became more obvious. However, the conformation of oligonucleotides with d(TA)(n) inserts changed a little in the presence of Zα1α2, and Zα1α2 had no effect on conformational transition of oligonucleotides with non-d(GC) or non-d(TA) inserts. Gel mobility shift assays further showed that Zα1α2 could bind to oligonucleotide with d(GC)(10). In other words, Zα1α2 can turn oligonucleotides with d(GC)(n) inserts into Z-DNA conformation and bind to it with high affinity.

Formation of a Snf1-Mec1-Atg1 Module on Mitochondria Governs Energy Deprivation-Induced Autophagy by Regulating Mitochondrial Respiration.

Autophagy is essential for maintaining glucose homeostasis, but the mechanism by which energy deprivation activates autophagy is not fully understood. We show that Mec1/ATR, a member of the DNA damage response pathway, is essential for glucose starvation-induced autophagy. Mec1, Atg13, Atg1, and the energy-sensing kinase Snf1 are recruited to mitochondria shortly after glucose starvation. Mec1 is recruited through the adaptor protein Ggc1. Snf1 phosphorylates Mec1 on the mitochondrial surface, leading to recruitment of Atg1 to mitochondria. Furthermore, the Snf1-mediated Mec1 phosphorylation and mitochondrial recruitment of Atg1 are essential for maintaining mitochondrial respiration during glucose starvation, and active mitochondrial respiration is required for energy deprivation-activated autophagy. Thus, formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration.