Mild overexpression of Atg14 increases autophagic activity in yeast [32
]. In mammals, overexpression of Barkor/Atg14(L) enhances autophagic activity even under nutrient-rich conditions [36
]. Thus, Atg14 seems to be one of the limiting factors regulating autophagic activity.
Autophagic activity is reduced in yeast cells expressing an Atg14 variant lacking the C-terminal half (hereafter, Atg14-ΔC) compared to cells expressing the full-length Atg14 (Atg14-FL). Cells expressing the Atg14-ΔC variant accumulate smaller autophagic bodies, indicating that Atg14 has a close relationship with the size of the autophagosome [32
]. We performed electron microscopy and measured the diameter of the smaller autophagic bodies accumulated in Atg14-ΔC cells (). The average diameter of autophagic bodies accumulated in Atg14-ΔC cells is approximately 66% of that in cells expressing Atg14-FL. Thus, the volume of each autophagic body in Atg14-ΔC cells is estimated to be 29% (the cube of 66%) of that in Atg14-FL cells. Autophagic activity is roughly proportional to the volume of autophagic bodies. Consistent with the estimation based on this electron microscopy, the actual autophagic activity in Atg14-ΔC cells, measured by an established biochemical assay, is approximately 33% of that in Atg14-FL cells [32
]. Thus, the C-terminal half of Atg14 is likely to be required to form a normal-sized autophagosome rather than to regulate the number of autophagosomes. How Atg14 regulates the size of autophagosomes is currently unknown. It is possible that the C-terminal half of Atg14 is directly involved in the modulation of autophagosome size. In this sense, it would be interesting to examine whether the amphiphilic helix within the C-terminal half is involved in modulating the curvature of the isolation membrane. Alternatively, the C-terminal half of Atg14 may regulate autophagosome size indirectly through one or more downstream molecules. Deletion of ATG14
affects the localization of Atg8, the Atg12-Atg5-Atg16 complex, and the Atg2-Atg18 complex [41
]. Smaller autophagic bodies are accumulated in cells expressing Atg8 variants with reduced activity [42
]. Similarly, the size of the autophagosome correlates with the protein levels of Atg8 [43
]. Thus, it is possible that Atg14 regulates autophagosome size indirectly through modulating Atg8 recruitment to the PAS.
Figure 3 Cells expressing the Atg14-ΔC variant accumulate smaller autophagic bodies. (a) Electron micrographs of autophagic bodies accumulated in BJ2168 cells lacking vacuolar proteases. Cells were cultured for 3.5h in nitrogen-depleted medium, (more ...)
As mentioned above, overexpression of Barkor/Atg14(L) activates autophagy in mammalian cells even under nutrient-rich conditions [36
], indicating that Barkor/Atg14(L) is one of the key players regulating autophagic activity in mammals. Other subunits of PtdIns 3-kinase complexes are also involved in the regulation of autophagic activity. UVRAG positively regulates maturation of the endosome and the autophagosome. Conversely, Rubicon negatively regulates maturation of the endosome and the autophagosome by sequestering UVRAG from the class C-VPS/HOPS complex [34
]. Beclin 1 also plays an important role in regulating autophagy. Beclin 1 interacts with multiple proteins in addition to the core subunits of the PtdIns 3-kinase complexes. One of these, Ambra1, positively regulates autophagy and plays a crucial role in neural development [30
]. Beclin 1 also interacts with Bcl-2, an antiapoptotic protein, that is believed to regulate the balance between autophagy and apoptosis [29
]. Thus, Beclin 1 may serve as a platform upon which cellular signals converge and function to regulate the crosstalk of multiple processes, including autophagy. This function of regulating the balance of multiple cellular events has not been reported for yeast Vps30/Atg6, which implies that Beclin 1 has obtained this regulatory role during evolution.
In addition to generating PtdIns(3)P
by the autophagy-specific PtdIns 3-kinase, dephosphorylation of PtdIns(3)P
also plays an important role in regulating autophagy in mammals. Overexpression of PtdIns(3)P
phosphatases decreases autophagic activity, while the knockdown or the expression of a dominant-negative form of the phosphatases enhances autophagy [45
]. It is currently unclear whether such regulation also occurs in yeast.
Taken together, Atg14 regulates autophagic activity, at least partially, both in yeast and mammals. However, the Barkor/Atg14(L)-containing PtdIns 3-kinase complex seems to play a more crucial role in determining autophagic activity than yeast Complex I, and the regulation of the Barkor/Atg14(L) complex may have evolved to function in a more sophisticated manner.