PDF(Pubmed)
Abstract：
The formation of autophagosomes mediating the sequestration of cytoplasmic materials is the central step of autophagy. Several phosphoinositides, which are signaling molecules on the membrane, are involved in autophagy. However, it is not always clear whether these phosphoinositides act directly at the site of autophagosome formation, or indirectly via the regulation of other steps or pathways. To address this question, we used a set of phosphoinositide probes to systematically examine their potential presence on autophagosomal membranes in yeast (Saccharomyces cerevisiae). We verified the specificity of these probes using mutant cells deficient in the production of the corresponding phosphoinositides. We then examined starved yeast cells co-expressing a phosphoinositide probe together with an autophagosomal membrane marker, 2Katushka2S-Atg8. Our data revealed that PtdIns(4,5)P2 and PtdIns(3,5)P2 were mainly present on the plasma membrane and vacuolar membrane, respectively. We observed only occasional co-localization between the PtdIns(4)P probe and Atg8, some of which may represent the transient passage of a PtdIns(4)P-containing structure near the autophagosomal membrane. In contrast, substantial colocalization of the PtdIns(3)P probe with Atg8 was observed. Taken together, our data indicate that only PtdIns(3)P is present in a substantial amount on the autophagosomal membrane. For other phosphoinositides involved in autophagy, either their presence on the autophagosomal membrane is very transient, or they act on other cellular membranes to regulate autophagy.
摘要：
自噬体的形成介导细胞质物质的螯合是自噬的核心步骤。几种磷酸肌醇，它们是膜上的信号分子，参与自噬。然而,尚不清楚这些磷酸肌醇是否直接作用于自噬体形成的位点，或间接通过调节其他步骤或途径。为了解决这个问题，我们使用了一组磷酸肌醇探针来系统地检查它们在酵母（酿酒酵母）自噬细胞膜上的潜在存在。我们使用在相应的磷酸肌醇产生中缺乏的突变细胞验证了这些探针的特异性。然后，我们检查了饥饿的酵母细胞共表达磷酸肌醇探针和自噬体膜标记物，2Katushka2S-Atg8。我们的数据显示，PtdIns（4,5）P2和PtdIns（3,5）P2主要存在于质膜和液泡膜上，分别。我们仅观察到PtdIns（4）P探针和Atg8之间的偶然共定位，其中一些可能代表自噬小体膜附近的含PtdIns（4）P结构的瞬时通过。相比之下,观察到PtdIns（3）P探针与Atg8的大量共定位。一起来看,我们的数据表明，只有PtdIns（3）P大量存在于自噬体膜上。对于参与自噬的其他磷酸肌醇，它们在自噬体膜上的存在是非常短暂的，或者它们作用于其他细胞膜来调节自噬。
