A research team led by Prof. Guo Yusong, an Associate Professor at The Hong Kong University of Science and Technology (HKUST), has made a pivotal discovery in understanding cellular protein transport mechanisms. Their findings, detailed in the Proceedings of the National Academy of Sciences (PNAS), reveal crucial insights into how cells transport proteins, a process essential for maintaining cellular function and implicated in various hereditary diseases.
Utilizing an innovative vesicle proteomics platform, the researchers systematically identified new cargo proteins and key accessory factors associated with two vital cellular transport complexes, known as AP-1 and AP-4. This research combines advanced vesicle reconstitution techniques with quantitative mass spectrometry to create a comprehensive map of previously unknown cargo proteins and regulatory factors.
The secretory pathway functions as a cellular postal service, ensuring proteins are delivered accurately to their intended destinations. Disruptions in this process can lead to severe physiological defects. “For years, the field has struggled to comprehensively map the cargo repertoire of adaptor complexes like AP-1 and AP-4, whose malfunctions are directly linked to serious human conditions such as MEDNIK syndrome, X-linked intellectual disability, and AP-4 deficiency syndrome,” stated Prof. Guo. Despite ongoing research, the complete list of proteins transported by these complexes has remained elusive.
This study marks a significant advancement in the field. “Our approach of reconstituting the transport process in vitro and applying quantitative proteomics allows us to directly identify which proteins require AP-1 or AP-4 for packaging into vesicles,” Prof. Guo continued. “This method has transitioned our understanding from a fragmented view to a more holistic perspective of their cargo landscape, revealing both new clients and the unexpected cellular machinery that AP-4 relies on.”
Innovative Techniques and Key Findings
The research team’s method integrates in vitro vesicle reconstitution with quantitative mass spectrometry, enabling the creation of transport vesicles in a controlled laboratory setting. This technique facilitates an exhaustive analysis of protein composition, shedding light on the specific cargo proteins that depend on AP-1 or AP-4 for transport from the trans-Golgi network, a central sorting hub within the cell.
Notably, the study identified the protein CAB45 as an AP-1-dependent cargo and discovered ATRAP as a novel cargo for AP-4. A major revelation addresses a long-standing question regarding how AP-4 can form transport vesicles in the absence of the well-known protein clathrin. The research uncovered that two cytosolic factors, WDR44 and PRRC1, are critical accessories for AP-4-mediated trafficking. When these factors were depleted, essential AP-4 cargoes, including ATG9A and ATRAP, were unable to exit their organelles, resulting in disruptions in vital cellular processes such as autophagy.
“Our findings not only reveal new cargo clients and essential co-factors for AP-1 and AP-4 but also provide a powerful toolkit for the scientific community to dissect the mechanisms of vesicular trafficking,” Prof. Guo emphasized. “This research opens new avenues for investigating the pathological mechanisms behind related diseases and potentially identifying new therapeutic targets.”
The collaboration between HKUST and The Hong Kong Polytechnic University (PolyU) was instrumental in this research. The co-corresponding authors of the study are Prof. Guo Yusong from HKUST and Prof. Yao Zhong-Ping from PolyU. The first author of the study is Dr. Peng Ziqing, a postdoctoral researcher at HKUST.
This breakthrough not only enhances our understanding of cellular transport mechanisms but also highlights the potential for future research aimed at addressing genetic disorders linked to malfunctions within these critical systems.
