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Mechanisms of secretory cargo sorting at the trans-Golgi network
Mechanisms of secretory cargo sorting at the trans-Golgi network
Protein sorting, processing and trafficking are central processes that maintain cellular functions, migration and homeostasis, ultimately defining health or disease of the organism. The secretory pathway comprises the compartments where these processes are realized. In this thesis the focus lies in particular on the last station of the Golgi apparatus (GA) and main sorting hub - the trans-Golgi network (TGN). There, proteins that have been modified post-translationally in earlier Golgi compartments, are sorted by their features, such as a transmembrane domain or a tag via certain receptors to intracellular destinations (e.g. endosomes, lysosomes) or for secretion (to apical and basolateral plasma membrane). Importantly, not all soluble secretory proteins are known to be recognized by a certain receptor, however, they are still sorted specifically to their final destinations. One mechanism involving the Ca2+-binding protein Cab45 was reported to facilitate the sorting of some secretory soluble proteins in the TGN (von Blume et al., 2011, 2012; Crevenna et al., 2016; Deng et al., 2018, Hecht et al., 2020): Ca2+-influx by secretory Ca2+- ATPase (SPCA1) in the TGN lumen after activation via actin/cofilin, leads to oligomerization of Cab45, which can bind its cargo (client) proteins and form Cab45-client-complexes that cluster and remain in the TGN lumen. Upon phosphorylation by Golgi kinase Fam20C the clusters are broken down for packaging into SM-rich vesicles destined for secretion. However, how these Cab45-client complexes are recognized for packaging into specific vesicles is unclear. In this thesis one more component is added to the machinery, the transmembrane protein TGN46, found in proximity-labelling approaches (BioID, APEX ; Deng et al., 2018), and shown here to be involved in the sorting of Cab45 client Lysozyme C (LyzC) by possibly indirect interaction with Cab45. Depletion of TGN46 indicates a delayed formation of post-Golgi vesicles containing LyzC in Retention-using-selective-hooks (RUSH)-based experiments and missorting by reduced co-localization with sphingomyelin (SM)-rich vesicles. Results that are similar to the phenotypes described previously for dysfunctional Cab45 and depletion of other components of the Cab45-dependent sorting mechanism (Deng et al., 2016; Hecht et al., 2020). In addition to this secretion-delayed phenotype of Cab45 clients upon deletion of TGN46, a novel hypersecretion of lysosomal hydrolases was observed with depletion of Cab45 in secretome analysis with subsequent mass spectrometric analysis. Secretion assays confirmed the influence of Cab45 depletion on increased secretion of prosaposin (PSAP), progranulin (PGRN) and with lesser degree Cathepsin D (CatD) implying a new role of Cab45 in sorting of lysosomal proteins. Secretion assays using cells with loss of lysosomal protein receptor sortilin and cation-independent mannose-6-phosphate receptor (M6PR) and Cab45 siRNA treatment indicated an influence of Cab45 on receptor-cargo interactions necessary for correct sorting. This hypothesis was substantiated by RUSH-based live-cell imaging observing the TGN export of PSAP in Cab45 knockout, -Ca2+-binding mutant and -wt rescue compared to control cells. Additionally, lysosomal positioning and co-localization of PSAP with lysosomes was altered with Cab45 deletion. The study gives evidence for the herein new proposed role of Cab45 in receptor-dependent lysosomal sorting in addition to the already reported receptor-independent sorting of soluble secretory proteins, which is extended with TGN46 as a new component.
Golgi Apparatus, Secretion, Protein sorting, trans-Golgi network, Cab45, TGN46
Tran, Mai Ly
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Tran, Mai Ly (2022): Mechanisms of secretory cargo sorting at the trans-Golgi network. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Protein sorting, processing and trafficking are central processes that maintain cellular functions, migration and homeostasis, ultimately defining health or disease of the organism. The secretory pathway comprises the compartments where these processes are realized. In this thesis the focus lies in particular on the last station of the Golgi apparatus (GA) and main sorting hub - the trans-Golgi network (TGN). There, proteins that have been modified post-translationally in earlier Golgi compartments, are sorted by their features, such as a transmembrane domain or a tag via certain receptors to intracellular destinations (e.g. endosomes, lysosomes) or for secretion (to apical and basolateral plasma membrane). Importantly, not all soluble secretory proteins are known to be recognized by a certain receptor, however, they are still sorted specifically to their final destinations. One mechanism involving the Ca2+-binding protein Cab45 was reported to facilitate the sorting of some secretory soluble proteins in the TGN (von Blume et al., 2011, 2012; Crevenna et al., 2016; Deng et al., 2018, Hecht et al., 2020): Ca2+-influx by secretory Ca2+- ATPase (SPCA1) in the TGN lumen after activation via actin/cofilin, leads to oligomerization of Cab45, which can bind its cargo (client) proteins and form Cab45-client-complexes that cluster and remain in the TGN lumen. Upon phosphorylation by Golgi kinase Fam20C the clusters are broken down for packaging into SM-rich vesicles destined for secretion. However, how these Cab45-client complexes are recognized for packaging into specific vesicles is unclear. In this thesis one more component is added to the machinery, the transmembrane protein TGN46, found in proximity-labelling approaches (BioID, APEX ; Deng et al., 2018), and shown here to be involved in the sorting of Cab45 client Lysozyme C (LyzC) by possibly indirect interaction with Cab45. Depletion of TGN46 indicates a delayed formation of post-Golgi vesicles containing LyzC in Retention-using-selective-hooks (RUSH)-based experiments and missorting by reduced co-localization with sphingomyelin (SM)-rich vesicles. Results that are similar to the phenotypes described previously for dysfunctional Cab45 and depletion of other components of the Cab45-dependent sorting mechanism (Deng et al., 2016; Hecht et al., 2020). In addition to this secretion-delayed phenotype of Cab45 clients upon deletion of TGN46, a novel hypersecretion of lysosomal hydrolases was observed with depletion of Cab45 in secretome analysis with subsequent mass spectrometric analysis. Secretion assays confirmed the influence of Cab45 depletion on increased secretion of prosaposin (PSAP), progranulin (PGRN) and with lesser degree Cathepsin D (CatD) implying a new role of Cab45 in sorting of lysosomal proteins. Secretion assays using cells with loss of lysosomal protein receptor sortilin and cation-independent mannose-6-phosphate receptor (M6PR) and Cab45 siRNA treatment indicated an influence of Cab45 on receptor-cargo interactions necessary for correct sorting. This hypothesis was substantiated by RUSH-based live-cell imaging observing the TGN export of PSAP in Cab45 knockout, -Ca2+-binding mutant and -wt rescue compared to control cells. Additionally, lysosomal positioning and co-localization of PSAP with lysosomes was altered with Cab45 deletion. The study gives evidence for the herein new proposed role of Cab45 in receptor-dependent lysosomal sorting in addition to the already reported receptor-independent sorting of soluble secretory proteins, which is extended with TGN46 as a new component.