(This article was originally published on EurekAlert! on October 22, 2025)

The secretory pathway in eukaryotic cells is crucial for maintaining cellular function and physiological activities, as it ensures the accurate transport of proteins to specific subcellular locations or for secretion outside the cell. A research team led by Prof. GUO Yusong from the Division of Life Science at the Hong Kong University of Science and Technology (HKUST) has been extensively investigating the molecular mechanisms by which cargo proteins are recognized and loaded into transport vesicles in the secretory pathway. The team has successfully reconstituted the packaging of multiple disease-related cargo proteins into vesicles along the secretory route, providing a powerful tool for dissecting the molecular mechanisms of cargo loading. In addition, they developed an innovative analysis platform that integrates vesicle reconstitution with electron microscopy and proteomics, enabling systematic identification of vesicle protein composition and morphological features. This comprehensive approach has proven effective in uncovering novel cargo clients and cellular factors that mediate vesicular trafficking (Figure 1).

Figure 1: Schematic of the research approach based on vesicle reconstitution combined with electron microscopy and proteomics. (References: PNAS 2019, PNAS 2021, PNAS 2025, Nature Plants 2021)

The research team led by Prof. Guo, in collaboration with Prof. YAO Zhong-Ping’s team at The Hong Kong Polytechnic University (PolyU), published their findings in PNAS. This study integrates in vitro vesicle reconstitution with quantitative mass spectrometry to systematically identify transport cargos mediated by the adaptor protein complexes AP-1 and AP-4. It also elucidates key cytosolic regulatory factors involved in AP-4–mediated vesicle trafficking.

Within the secretory pathway, the trans-Golgi network (TGN) serves as a central sorting hub, packaging proteins precisely into transport vesicles. Sorting errors can prevent cargos from reaching their correct targets, leading to defects in cell polarity, immune function, and other physiological processes. Key factors involved in protein sorting at the TGN are adaptor protein (AP) complexes, AP-1 and AP-4. Mutations in their genes are closely linked to several human diseases: AP1S1 mutations (encoding the σ1 subunit of AP-1) are associated with MEDNIK syndrome; AP1S2 mutations can cause X-linked intellectual disability; and mutations in any AP-4 subunit lead to “AP-4 deficiency syndrome,” a complex hereditary spastic paraplegia. Therefore, defining the cargo clients of AP-1 and AP-4 is crucial for understanding their physiological and pathological roles. However, the full repertoire of cargos and cofactors they mediate remains unclear. Notably, AP-4–mediated TGN export appears to be clathrin-independent, suggesting that vesicle formation may depend on yet-unidentified accessory factors.

In this study, Prof. Guo’s team performed vesicle formation assays using AP1γ1 or AP4ε knockout cells, isolated vesicle fractions, and conducted proteome-wide analysis by quantitative mass spectrometry. This approach allowed them to identify AP-1– and AP-4–dependent cargo proteins exported from the TGN, as well as key cytosolic accessory factors required for AP-4–mediated transport. Subsequent biochemical validation revealed that CAB45 is an AP-1–dependent cargo, while ATRAP (an angiotensin II type I receptor–associated protein) is an AP-4–dependent cargo. Notably, AP-4 recognizes a tyrosine-based motif at the cytosolic terminus of ATRAP to mediate its loading into transport vesicles from the Golgi.

The study also found that the cytosolic proteins WDR44 and PRRC1 play critical roles in AP-4–mediated cargo sorting. When WDR44 levels were knocked down, the AP-4 cargo ATG9A abnormally accumulated at the Golgi. Similarly, PRRC1 knockout caused ATG9A to be retained in the endoplasmic reticulum, impairing cellular autophagy.  Another AP-4 cargo, ATRAP, also accumulated in the Golgi under these conditions. “These results not only deepen our understanding of AP-1 and AP-4 functions in secretory trafficking but also provide a powerful methodological toolkit for systematically dissecting the mechanisms of specific accessory factors,” said Prof. Guo.

The study’s co-corresponding authors are Prof. Guo of HKUST and Prof. Yao of PolyU. Dr. PENG Ziqing, a postdoctoral researcher at HKUST, is the first author.

Prof. Guo (second right in the front row) and his research team.