"Trk1,Trk2,Hal4及Hal5調控細胞自噬之角色"

Abstract

細胞自噬是一個蛋白質分解機制，藉由雙層膜構造-自噬小體吞噬細胞質內不必要之物質，包含蛋白質或損壞的胞器，進而與液泡或溶酶體結合後水解。這個高度保守的機制有維持細胞內滲透壓恆定的重要功能，細胞自噬一旦失調即會導致許多人類疾病的產生，例如神經退化性疾病和癌症。當細胞受到外界環境刺激或接受到養分或氧氣不足時，就會啟動細胞自噬。此過程可分為四個階段，細胞自噬啟動、分隔膜形成、自噬小泡成熟和溶酶體分解。每一個階段都有許多特定蛋白調控，且許多磷酸酶也參與其中。前人研究發現Hal4和Hal5在維持細胞膜上運輸蛋白的穩定性扮演重要角色。這兩個功能相似的磷酸酶藉由調控鉀離子運輸蛋白Trk1-Trk2和穩定細胞膜上Trk1，促進鉀的攝取而調控鹽類耐受性。此外，我們先前研究證實剔除hal4和hal5基因的酵母菌會使細胞自噬活性下降且降低細胞生長速率。藉由補充鉀離子或是過度表現蛋白，可以恢復細胞生長速率，但只能有限地修復細胞自噬活性。本研究致力於研究是否Trk1和Trk2運輸蛋白也參與細胞自噬的調控。然而，根據我的實驗結果，發現剔除Trk1和Trk2 基因的酵母菌所產生的細胞自噬活性下降和生長速率遲緩，皆可藉由補充鉀離子全然恢復，證實Trk1和Trk2與細胞自噬的調控沒有直接關係。

Autophagy is a protein degradation pathway by engulfing cytoplasmic components, including protein aggregates and damaged organelles, into autophagosomes that later fuse with the vacuole in yeast or the equivalent organelle lysosome in metazoans. It is a highly conserved process occurring from yeast to mammals and is critical to maintaining cell homeostasis. Deregulated autophagy is involved in various human diseases, such as neurodegenerative diseases, cancers, and metabolic diseases. When cells suffer stress such as starvation or heat, autophagy will be induced. This process can be divided into four stages: initiation, autophagosome formation, autophagosome maturation, and lysosome degradation. Each stage is regulated by specific proteins and several kinases are also involved. Hal4 and Hal5 are two functionally redundant Ser/Thr protein kinases involved in salt tolerance. Previous studies had shown the important role of Hal4 and Hal5 protein kinases to maintain the stability of plasma membrane transporter protein Trk1 and Trk2. Hal4 and Hal5 promote K+ uptake by the potassium transporter Trk1-Trk2 and stabilize Trk1 in the plasma membrane. A previous study from our laboratory had found that knockout of HAL4 and HAL5 genes in budding yeast caused severe autophagy defects and slower growth phenotype. Supplementation of potassium and overexpression of Npr1 can rescue the growth defect but not autophagy defect in the double knockout mutant cells. In this study, I aim to investigate whether Trk1 and Trk2 are also involved in autophagy regulation. My experimental results show that the autophagy and growth defect in the double knockout trk1trk2 mutant can be rescued by potassium supplementation completely, which demonstrates the potassium transporter protein Trk1 and Trk2 are not directly involved in autophagy regulation.