Molecular Characterization of Autophagy

真核细胞通过合成和降解来改变它们的蛋白质补体以适应环境的变化。许多人都知道，许多实验室正在研究营养适应和胁迫过程中转录和翻译的调节。然而，降解去除多余或受损蛋白质的分子机制尚未解决。众所周知，哺乳动物细胞通过一种称为自噬的过程隔离蛋白质和细胞器以降解溶酶体，从而适应氨基酸或血清饥饿。考虑到操纵哺乳动物遗传学的难度，自噬的分子事件还没有被定义。邓恩博士的实验室开发了一种酵母菌模型--毕赤酵母，用于研究过氧化物酶体内的自噬和溶酶体蛋白分解。在这种酵母中，两种形态上截然不同的自噬--微型自噬和宏观自噬--可以受到营养因素的调节。巴氏假单胞菌模型为研究过氧化物体降解的分子事件提供了最好的机会，因为过氧化物体的降解速度很快，而且不能降解过氧化物体的突变株可以很容易地通过灵敏的比色直接菌落分析来识别。有人认为，葡萄糖诱导的过氧化体选择性自噬通过四个事件进行：葡萄糖信号；早期隔离事件，包括过氧化物酶体识别和液泡膜内陷；晚期隔离事件，包括同型膜融合；和空泡降解。邓恩已经确定了八个葡萄糖诱导的选择性微自噬突变体(GSAL-gsa8)，它们在液泡降解的上游事件中存在缺陷。该项目的第一个目的是研究自噬的分子特征，包括GSA基因的鉴定和GSA基因产物的亚细胞定位。首先，Dunn将识别和表征三个不同事件所需的基因，GSA1(葡萄糖信号事件)，GSA4(过氧化体识别)和GSA7(液泡膜的同型融合)；GSA1和GSA7已经被克隆和测序。然后，Dunn将利用细胞、分子和遗传方法来定义功能结构域和基序，并识别GSAI p、GSA4p和GSA7p的相互作用蛋白。人们期望这些研究将提供对这些蛋白质在过氧化物体自噬中的功能作用的见解。Dunn的工作假设是，GSAlp、GSA4p和GSA7p是葡萄糖适应过程中过氧化体降解的微观自噬途径的三个不同事件所需的功能独特的蛋白质。GSA4将按照已成功用于识别GSA1和GSA7的程序进行克隆和测序。GSA4p将通过比较gsa4-1和Delta-gsa4(即缺失)突变体的表型来验证。然后，GSAlp、GSA4p和GSA7p在甲醇和葡萄糖适应细胞中的亚细胞位置将首先在酵母中表达标记为GSAp的HA表位，然后对固定细胞进行免疫定位，并使用HA表位抗体对特定的亚细胞部分进行免疫印迹。首先，这些蛋白质的最小功能单位将通过缺失分析来确定。然后，可能参与自噬的最小功能单位(即酶活性部位、蛋白结合基序、磷酸化和肉豆蔻化部位)内的假定功能结构域将发生突变，并将评估突变的GSA蛋白拯救Delta-GSA表型的能力。其次，为了更好地确定具有自噬活性的氨基酸，我们将对GSAp进行随机PCR突变，克隆不能挽救Delta-GSA表型的突变的GSAp，并对突变进行测序。最后，这些蛋白质的候选分子将通过双杂交和“高表达”抑制子分析来确定，并通过免疫共沉淀进行验证。

Eukaryotic cells adapt to environmental changes by altering their protein complements through synthesis and degradation. Much is known about, and many laboratories are investigating, the regulation of transcription and translation during nutrient adaptation and stress. However, the molecular mechanisms responsible for the degradative removal of superfluous or damaged proteins have not been resolved. It is well known that mammalian cells adapt to amino acid or serum starvation by sequestering proteins and organelles for lysosomal degradation via a process called autophagy. Given the difficulty in manipulating mammalian genetics, the molecular events of autophagy have yet to be defined. Dr. Dunn's laboratory has developed a yeast model, Pichia pastoris, for studying autophagy and lysosomal proteolysis of peroxisomes. Two morphologically distinct kinds of autophagy, microautophagy and macroautophagy, can be regulated in this yeast by nutritional factors. The P. pastoris model offers arguably the best opportunity to investigate the molecular events of peroxisome degradation because the degradation is rapid and because mutants unable to degrade peroxisomes can be easily identified by a sensitive colorometric direct colony assay. It is proposed that glucose-induced selective autophagy of peroxisomes proceeds through four events: glucose signalling; early sequestration events, including peroxisome recognition and vacuole membrane invaginations; late sequestration events, including homotypic membrane fusion; and vacuolar degradation. Dunn has identified eight glucose-induced selective microautophagy mutants (gsal- gsa8) which are defective in the events upstream of vacuolar degradation. The first aim of this project is a molecular characterization of autophagy involving the identification of the GSA genes and the subcellular location of the GSA gene products. Initially, Dunn will identify and characterize three genes that are required for three different events, GSA1 (glucose signalling event), GSA4 (peroxisome recognition), and GSA7 (homotypic fusion of the vacuolar membrane); GSAl and GSA7 have already been cloned and sequenced. Dunn will then utilize cellular, molecular, and genetic approaches to define functional domains and motifs and to identify interacting proteins of GSAI p, GSA4p, and GSA7p. The expectation is that these studies will provide insights into the functional roles of these proteins in peroxisome autophagy. Dunn's working hypothesis is that GSAlp, GSA4p, and GSA7p are functionally unique proteins required for three different events of the micro-autophagy pathway of degradation of peroxisomes during glucose adaptation. GSA4 will be cloned and sequenced following procedures that have been used successfully to identify GSA1 and GSA7. The GSA4p will be verified by comparing the phenotypes of gsa4-1 and delta-gsa4 (i.e., deletion) mutants. Then, the subcellular locations of GSAlp, GSA4p, and GSA7p will be determined in methanol- and glucose-adapting cells by first expressing an HA-epitope tagged GSAp in the yeast followed by immunolocalization of fixed cells and Western blotting of specific subcellular fractions using an antibody to the HA-epitope.The functional motifs and molecular associates of these proteins will be characterized. First, the minimal functional unit of each of these proteins will be defined by deletion analysis. Putative functional domains within the minimal functional unit (i.e., enzymatic active sites, protein binding motifs, and phosphorylation and myristylation sites) that may be involved in autophagy will then be mutated and the ability of the mutated gsa protein to rescue the delta-gsa phenotype will be evaluated. Second, in order to better define the amino acid (or amino acids) that confers autophagy activity, we will perform random PCR mutagenesis of the GSAp, clone the mutated GSAp by its inability to rescue the delta-gsa phenotype, and sequence the mutation. Finally, candidates for molecular associates of these proteins will be identified by two-hybrid and "high-expression" suppressor analyses and verified by co-immunoprecipitation.