The Ccl1-Kin28 Kinase Complex Regulates Autophagy Under Nitrogen Starvation

Under the condition of nutritional deprivation or other cell stress(high temperature or oxygen deficiency etc.), cytosol, long-lived protein and damaged-organelles cell are sequestered into the double-membrane autophagosome, and then are transferred into yeast vacuole(or lysosome in mammalian) for degradation. Regenerated nutrients are sent back to the cytosol for further utilization to maintain cell survival. Yet the excess autophagy would damage cell homeostasis and cause cell death, it is necessary to well demonstrate the mechanism of autophagy,which would provide powerful theoretical basis for diagnosis and treatment of disease.By screening of kown kinase and their regulation factor,we identified Ccl1-Kin28 kinase complex(homologue in mammalian is Cyclin H-Cdk7)which fuctions mainly in RNA polymerase II transcription. Autophagy is completely blocked by depletion the subunits of Ccl1-Kin28 complex, and Cvt pathway is partially affected. Ccl1 is a cyclin which promote autophagy by maintaining the kinase activity of Kin28. Meanwhile Ccl1 itself is also under the control of proteasome pathway. During the prolong starvation, Ccl1 is gradually degraded by the proteasome and blocks the autophagy activity. When overexpression of Ccl1 inhibits degradation,Autophagy activity is consequently increased. That is, there exists feedback regulation machnism in cell. Ccl1 which connect the two important degradation system in cell, by regulation of Proteasome on the protein level to adjust the autophagy activity to most suitable conditon. At the time of screen of factors regulating the ubiquitin process, we found that the E1 and E2 enzyme regulating Ccl1 degradation was Uba1 and Ubc3/Cdc34, respectively.In addition, we made systemic analysis to Atg protein by fluorescence microscope and identified that Ccl1 involved in the early stage of autophagy regualtion, mainly influencing the assembly of Atg1 complex. After inactivation of Ccl1 or Kin28, the protein level of Atg29 and Atg31 reduce dramatically. In conclusion, Atg29 and Atg31 are the important effector of Ccl1-Kin28 in autophagy regulation.