Molecular Characterization of Autophagy

真核细胞通过合成和降解改变其蛋白质补体来适应环境变化。营养适应和胁迫过程中转录和翻译的调控已经有了很多了解，许多实验室也在进行研究。然而，负责降解去除多余或受损蛋白质的分子机制尚未得到解决。众所周知，哺乳动物细胞通过隔离蛋白质和细胞器以经由称为自噬的过程进行溶酶体降解来适应氨基酸或血清饥饿。鉴于操纵哺乳动物遗传学的困难，自噬的分子事件尚未被定义。Dunn博士的实验室已经开发了一种酵母模型Pichia pastoris，用于研究过氧化物酶体的自噬和溶酶体蛋白水解。两种形态上不同的自噬，微自噬和大自噬，可以在这种酵母中调节营养因素。巴斯德毕赤酵母模型可以说是研究过氧化物酶体降解的分子事件的最佳机会，因为降解是快速的，并且因为不能降解过氧化物酶体的突变体可以通过灵敏的比色直接菌落测定容易地鉴定。有人提出，葡萄糖诱导的过氧化物酶体的选择性自噬通过四个事件进行：葡萄糖信号传导;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;和液泡降解。Dunn已经鉴定了八种葡萄糖诱导的选择性微自噬突变体（gsa 1-gsa 8），其在液泡降解的上游事件中有缺陷。本项目的第一个目的是对自噬进行分子表征，包括GSA基因的鉴定和GSA基因产物的亚细胞定位。 最初，Dunn将鉴定和表征三种不同事件所需的三种基因，GSA 1（葡萄糖信号事件），GSA 4（过氧化物酶体识别）和GSA 7（液泡膜的同型融合）; GSA 1和GSA 7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，并鉴定GSA 1 p、GSA 4p和GSA 7 p的相互作用蛋白。 期望这些研究将为这些蛋白质在过氧化物酶体自噬中的功能作用提供见解。Dunn的工作假设是GSA 1 p、GSA 4p和GSA 7 p是葡萄糖适应期间过氧化物酶体降解的微自噬途径的三种不同事件所需的功能独特的蛋白质。将按照已成功用于鉴定GSA 1和GSA 7的程序对GSA 4进行克隆和测序。将通过比较gsa 4 -1和delta-gsa 4的表型来验证GSA 4p（即，缺失）突变体。然后，通过首先在酵母中表达HA-表位标记的GSAp，然后通过固定细胞的免疫定位和使用HA-表位抗体的特异性亚细胞级分的Western印迹，将确定GSA 1 p、GSA 4p和GSA 7 p在甲醇和葡萄糖适应细胞中的亚细胞位置。 首先，这些蛋白质中的每一个的最小功能单元将通过缺失分析来定义。最小功能单位内的推定功能结构域（即，酶活性位点、蛋白结合基序、磷酸化和肉豆蔻酰化位点），然后将其突变，并评价突变的GSA蛋白拯救δ-GSA表型的能力。第二，为了更好地定义赋予自噬活性的氨基酸（或多种氨基酸），我们将对GSAp进行随机PCR诱变，通过其不能拯救δ-gsa表型来克隆突变的GSAp，并对突变进行测序。最后，这些蛋白质的分子伴侣的候选人将被确定的双杂交和“高表达”抑制分析和验证的免疫共沉淀。

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.