Hal4和Hal5對於細胞自噬途徑的調控

Abstract

細胞自噬是一個高度保守的機制，藉由雙層膜構造–自噬小體，來選擇性或是非選擇性地包裹細胞質中的物質，隨之自噬小體與液泡或是溶小體融合，由水解酵素將細胞質的成分分解。細胞自噬，需要被適當地調控以避免有害影響以及維持胞內恆定。前人研究中證實Hal4和Hal5對於維持運輸蛋白在細胞膜上的穩定性扮演重要角色，且由高通量的實驗中發現Hal5跟Atg31具有交互作用的關係，而在本研究中則發現，Hal4和Hal5具有調控細胞自噬的功能。我們在踢除hal4與hal5基因的酵母菌中，觀察到細胞自噬活性下降跟細胞生長嚴重遲緩，而藉由在培養液中添加鉀離子或是大量表現Npr1蛋白，則可以改善突變細胞的生長缺失，但是對自噬能力的修復作用有限。在養分缺失的環境中，hal4與hal5基因剔除的細胞執行細胞自噬的能力下降，且細胞中Atg1激酶複合體脫離自噬體前驅構造的效率也明顯減緩。根據我們的實驗結果，我們推測Hal4和Hal5蛋白激酶可能參與調控細胞自噬的晚期步驟。

Autophagy is a highly conserved pathway responsible for the degradation of cytoplasmic components. During autophagy, cargo is selectively or non-selectively engulfed by a unique double membrane structure referred to as an autophagosome and subsequently fused with the vacuole or lysosome. This pathway requires proper regulation to prevent deleterious effects and preserve homeostasis. Previous studies had proved the important role of the functionally redundant Hal4 (Sat4) and Hal5 protein kinases in the regulation of plasma membrane transporter stability. In addition, the interaction between Hal5 and Atg31 had been found by a high-throughput experiment. In this research, we discovered a new function for the functionally redundant protein kinases, Hal4 and Hal5, on general autophagy regulation. In budding yeast, chromosomal deletion of HAL4 and HAL5 genes causes server defects on autophagy activity and growth rate. By supplementing potassium or overexpressing Npr1, the growth, but not of the autophagy, defects of the mutant could be largely rescued. Simultaneous loss of Hal4 and Hal5 results in decreasing of autophagy activity under nutrient-depletion conditions and this result is associated with block of the Atg1 kinase complex release from the pre-autophagosomal structure (PAS). Our results suggest that Hal4 and Hal5 kinase proteins might regulate general autophagy through affecting the late stage of autophagy pathway.