First person – Xiuping Sun

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Xiuping Sun is first author on ‘A quantitative study of the Golgi retention of glycosyltransferases’, published in JCS. Xiuping conducted the research described in this article while a PhD candidate in Lu Lei's lab at School of Biological Sciences, Nanyang Technological University, Singapore. She is now a postdoc in the lab of Wang Jigang, Yang Qinhe at Shenzhen People's Hospital, Shenzhen, China investigating the roles of disease-related genes in neurodegenerative diseases.

A quantitative study of the Golgi retention of glycosyltransferases

How Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by the ectodomain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention but not retrieval should be the primary mechanism for their Golgi localization. We proposed to use the Golgi residence time to quantitatively and systematically study Golgi retention of glycosyltransferases. Various swapping chimeras between ST6GAL1 and either transferrin receptor or tumor necrosis factor α quantitatively revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, transmembrane domain, and ectodomain, to Golgi retention. We found that each of the three regions is sufficient to produce retention in an additive manner. The N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to the effect of the transmembrane domain. Therefore, the long N-terminal cytosolic tail and transmembrane domain can be a Golgi export signal for transmembrane secretory cargos.

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

The fidelity of Golgi glycosylation is, in part, ensured by compartmentalization of enzymes within the stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterization of the clientele of GOLPH3 and its paralogue GOLPH3L is lacking. GOLPH3’s role is of particular interest as it is frequently amplified in several solid tumor types. Here, we apply two orthogonal proteomic methods to identify GOLPH3+3L clients and find that they act in diverse glycosylation pathways or have other roles in the Golgi. Binding studies, bioinformatics, and a Golgi retention assay show that GOLPH3+3L bind the cytoplasmic tails of their clients through membrane-proximal positively charged residues. Furthermore, deletion of GOLPH3+3L causes multiple defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

Proteolytic processing of secretory pathway kinase Fam20C by site-1 protease promotes biomineralization

Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

A quantitative study of the Golgi retention of glycosyltransferases

ABSTRACTHow Golgi glycosyltransferases and glycosidases (hereafter glycosyltransferases) localize to the Golgi is still unclear. Here, we first investigated the post-Golgi trafficking of glycosyltransferases. We found that glycosyltransferases can escape the Golgi to the plasma membrane, where they are subsequently endocytosed to the endolysosome. Post-Golgi glycosyltransferases are probably degraded by the ecto-domain shedding. We discovered that most glycosyltransferases are not retrieved from post-Golgi sites, indicating that retention but not retrieval should be the main mechanism for their Golgi localization. We proposed to use the Golgi residence time to study the Golgi retention of glycosyltransferases quantitatively and systematically. Various chimeras between ST6GAL1 and either transferrin receptor or tumor necrosis factor α quantitatively revealed the contributions of three regions of ST6GAL1, namely the N-terminal cytosolic tail, transmembrane domain and ecto-domain, to the Golgi retention. We found that each of the three regions is sufficient to produce a retention in an additive manner. The N-terminal cytosolic tail length negatively affects the Golgi retention of ST6GAL1, similar to what is known of the transmembrane domain. Therefore, long N-terminal cytosolic tail and transmembrane domain can be a Golgi export signal for transmembrane secretory cargos.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.

Multiple features within Sed5p mediate it’s COPI-independent trafficking and Golgi localization

ABSTRACTProtein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi – the Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells a single syntaxin - the SNARE Sed5p - oversees membrane fusion within the Golgi. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the Golgi retention and trafficking of Sed5p between the Golgi and the ER is independent of COPI function, the composition of the transmembrane domain, and binding of the Sec1-Munc18 (SM) protein Sly1p. Rather, the steady state localization of Sed5p to the Golgi appears to be primarily conformation-based relying on intra-molecular associations between the Habc domain and SNARE-motif.

CCDC186 controls dense-core vesicle cargo sorting by exit

ABSTRACTThe regulated release of peptide hormones depends on their packaging into dense-core vesicles (DCVs). Two models have been proposed for DCV cargo sorting. The “sorting by entry” model proposes that DCV cargos selectively enter nascent DCVs at the trans-Golgi network (TGN). The “sorting by exit” model proposes that sorting occurs by the post-TGN removal of non-DCV cargos and retention of mature DCV cargos. Here we show that the coiled-coil protein CCDC186 controls sorting by exit. Ccdc186 KO insulinoma cells secrete less insulin, fail to retain insulin and carboxypeptidase E in mature DCVs at the cell periphery, and fail to remove carboxypeptidase D from immature DCVs. A mutation affecting the endosome-associated recycling protein (EARP) complex causes similar defects in DCV cargo retention and removal. CCDC186 and EARP may act together to control the post-Golgi retention of cargos in mature DCVs.