26S蛋白酶体是负责蛋白质降解的关键细胞器，对细胞功能至关重要，但其自身如何被调控还很不清楚。我们发现蛋白酶体的Rpt2亚基上存在三种化学修饰，即N端肉豆蔻酰化、丝氨酸磷酸化和酪氨酸磷酸化。首先，Rpt2的肉豆蔻酰化在进化中高度保守，很可能对于整个蛋白酶体在细胞膜上的定位至关重要。其次，Rpt2-Ser4的磷酸化会抑制Rpt2插膜，从而动态调控蛋白酶体的膜定位。第三，只有膜定位的Rpt2才会发生Tyr439位点的酪氨酸磷酸化。该位点对于蛋白酶体的组装非常重要，而负责该位点磷酸化的有可能是膜蛋白EGFR。综上，Rpt2亚基上这三种鲜有研究的修饰之间相互作用，决定了蛋白酶体的膜定位，并将膜上蛋白酶体的组装与细胞信号转导联系起来。我们将利用包括基因编辑、点击化学、定量质谱和动物模型在内的一系列手段，阐明上述蛋白酶体修饰的调控机制，解读蛋白酶体膜定位的生理功能及其在进化中的意义。

The 26S proteasome is an essential machinery for protein degradation in all eukaryotic cells. Despite its well established importance for cell viability, how this protein complex itself is regulated under various pathophysiological conditions remains largely unknown. We and others have provided initial evidence that chemical modifications (such as phosphorylation) play important roles in fine-tuning proteasome assembly, activity as well as intracellular localization. As such, proteasome regulation has gained increasing attention in recent years and is now positioned at the frontier of interdisciplinary research. In this Proposal, we focus on three types of modification that occur on a single subunit, Rpt2, of the 26S proteasome, including its N-terminal myristoylation, serine phosphorylation and tyrosine phosphorylation. Our preliminary data indicate that N-myristoylation, which is conserved from yeast to human, is critical for membrane anchoring of Rpt2 and probably of the whole proteasome. This effect is counterbalanced by phosphorylation of Ser4, a site that is conserved in vertebrates but not present in lower organisms. Membrane localization is also found to be a prerequisite for phosphorylation of Rpt2 at its penultimate residue, Tyr439, which is within the C-terminal HbYX motif critical for proteasome assembly. Phospho-proteomic studies have suggested that an oncogenic form of EGFR, a membrane protein, may be responsible for Rpt2-Y439 phosphorylation. Together, these conserved but rarely studied modifications may determine the localization of a select pool of 26S proteasomes, linking membrane proteasome assembly to cancer cell signaling. Here we propose a broad array of mechanistic and functional studies using canonical biochemical and cell biology approaches in conjunction with cutting-edge technology including gene editing, click chemistry, super-resolution microscopy, quantitative mass spectrometry and new animal models, in order to elucidate the molecular underpinnings of those proteasome modifications and the biological relevance of membrane-tethered proteasomes in health and disease and during evolution.