Trans-Golgi Network: Sorting secretory proteins
A receptor protein called TGN46 has an important role in sorting secretory proteins into vesicles going to different destinations inside cells.
- Department of Cell Biology, Yale University School of Medicine, United States
Approximately 14% of all protein-coding genes in humans produce proteins secreted by tissues and cells (Uhlén et al., 2019). These proteins control various essential processes, including immunity, metabolism and cellular communication. Secretory proteins also play significant roles in numerous diseases, such as neurological disorders and cancer (Brown et al., 2013).
Once a secretory protein has been synthesized in the endoplasmic reticulum, it must travel to another cellular compartment known as the trans-Golgi network before it can be released. The trans-Golgi network acts like a mail distribution centre, sorting secretory proteins into vesicles that travel to specific destinations, such as the cell surface, various compartments inside the cell (such as endosomes and lysosomes), or distinct domains in the plasma membrane (Figure 1A; Ford et al., 2021). A fundamental question in cell biology is how secretory proteins are effectively separated from one another and sorted into vesicles that will take the protein to the correct location.
Figure 1
Cargo sorting at the trans-Golgi network.
(A) In order for secretory proteins to reach their destination, they must first enter the endoplasmic reticulum (ER) and travel to the trans-Golgi network (TGN). Once there, proteins are sorted into different types of vesicles: constitutive vesicles (CV), clathrin-coated vesicles (CCV), the CARTS mentioned in the main text, or immature secretory granule (ISG). These vesicles carry a protein either to the plasma membrane (PM) to be immediately secreted, to mature secretory granules (MSG) to store it for future secretion, or to the lysosome (LYS) for degradation (B) Previous work showed that mannose-6-phosphate receptor (M6P-R, green) is responsible for sorting lysosomal enzymes (red small circles) into CCVs that are travelling to the lysosome. (C) Lujan et al. found that the transmembrane protein TGN46 (red) also acts as a cargo receptor and is responsible for sorting a secreted protein called PAUF (blue small circle) into CART vesicles destined for the cell surface. CARTS: CARriers TGN to the cell Surface; PAUF: pancreatic adenocarcinoma upregulated factor.
In the early 1980s, Stuart Kornfeld and colleagues provided the first mechanistic insight into protein sorting in the Golgi when they discovered a cargo receptor called M6P-R (short for mannose-6-phosphate receptor) within the membrane of the trans-Golgi network. This receptor can recognise and bind to a specific mannose-6-phosphate tag on lysosomal enzymes, and package them into vesicles destined for the lysosome (Dahms et al., 1989; Figure 1B).
This discovery raised the possibility that secretory proteins may be sorted by a similar receptor-ligand binding mechanism. However, the details of the sorting process remained a mystery. Now, in eLife, Felix Campelo (Barcelona Institute of Science and Technology) and colleagues – including Pablo Lujan as first author – report that a well-known protein called Transmembrane Network Protein 46 (TGN46) may be involved (Lujan et al., 2023).
TGN46 has long been hypothesized to carry out a sorting role in the trans-Golgi network as it displays many typical features for a cargo receptor. For instance, it rapidly cycles between the Golgi and plasma membrane, and the vesicles that transport it to the cell surface (known as CARTS) have been shown to carry secretory proteins, such as PAUF (short for pancreatic adenocarcinoma upregulated factor; Banting and Ponnambalam, 1997; McNamara et al., 2004). To investigate this hypothesis, Lujan et al. knocked out the gene for TGN46 from cells grown in a laboratory. This significantly reduced the amount of PAUF the cells secreted, suggesting that TGN46 acts as a cargo receptor for this secretory protein (Figure 1C).
Previous studies have shown that CARTS require an enzyme called protein kinase D in order to detach themselves from the membrane of the trans-Golgi network (Wakana et al., 2012). If this enzyme is inactivated and unable to perform this role, long tubules carrying cargo molecules will emerge from the Golgi membrane (Liljedahl et al., 2001). Lujan et al. found PAUF proteins in the tubules of wild-type cells but not in the tubules of the mutant cells lacking TGN46. This indicates that TGN46 is required to load PAUF into CART vesicles.
Next, Lujan et al. set out to find which parts of the TGN46 receptor were responsible for sorting and packaging secretory proteins. They found that the cytosolic domain of the receptor was not required for PAUF export, which is surprising given that adaptor proteins, which help to form vesicles, bind to other cargo receptors via this domain (Cross and Dodding, 2019). Changing the size of the transmembrane domain in TGN46 also did not impact the location of the receptor, as would be expected; instead it mildly affected how well PAUF was sorted into CARTS. Most importantly, Lujan et al. found that TGN46 only needs its luminal domain (the part of the receptor which faces the interior of the Golgi) in order to sufficiently sort PAUF proteins into CARTS.
This research sheds light on how secretory proteins are sorted into the correct vesicle at the trans-Golgi network. It also provides significant evidence that TGN46 acts as a cargo receptor, something which has been speculated in the field for over three decades. These exciting findings also raise new questions about how the luminal domain of TGN46 is able to recognize PAUF: is this recognition specific and based on ‘lock and key interactions’, or could it be based on the biophysical properties of the highly disordered luminal domain of TGN46?
It also remains to be seen which other secretory proteins TGN46 might be responsible for sorting. TGN46 is known to interact with another cell surface receptor called integrin beta 1, which is involved in cell adhesion at specific sites in the plasma membrane (Wang and Howell, 2000). This suggests that TGN46 may help to export proteins to specific domains at the cell surface. Future work investigating these questions, as well as others, will provide significant insights into the molecular mechanisms of protein secretion.
References
-
TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphologyBiochimica et Biophysica Acta 1355:209–217.https://doi.org/10.1016/s0167-4889(96)00146-2
-
The human secretome atlas initiative: implications in health and disease conditionsBiochimica et Biophysica Acta 1834:2454–2461.https://doi.org/10.1016/j.bbapap.2013.04.007
-
Motor-cargo adaptors at the organelle-cytoskeleton interfaceCurrent Opinion in Cell Biology 59:16–23.https://doi.org/10.1016/j.ceb.2019.02.010
-
Mannose 6-phosphate receptors and lysosomal enzyme targetingThe Journal of Biological Chemistry 264:12115–12118.
-
Cargo sorting at the trans-Golgi network at a glanceJournal of Cell Science 134:jcs259110.https://doi.org/10.1242/jcs.259110
-
Rapid dendritic transport of TGN38, a putative cargo receptorMolecular Brain Research 127:68–78.https://doi.org/10.1016/j.molbrainres.2004.05.013
Article and author information
Author details
Publication history
- Version of Record published: November 24, 2023 (version 1)
Copyright
© 2023, Parchure and von Blume
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 412
- Page views
-
- 75
- Downloads
-
- 0
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Trans-Golgi Network: Sorting secretory proteins
eLife 12:e93490.
https://doi.org/10.7554/eLife.93490
Further reading
-
- Cell Biology
Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson’s disease (PD) and Crohn’s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.
-
- Cell Biology
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.
