Segregation of the membrane cargoes, BACE1 and amyloid precursor protein (APP) throughout the Golgi apparatus.

The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.

The Golgi apparatus is a pivotal secretory organelle in membrane trafficking, a hub responsible for posttranslational modifications, sorting, and trafficking of newly synthetized proteins received from the endoplasmic reticulum (ER). Different protein cargoes have been shown to travel through the Golgi stacks with different kinetics. Dysregulated transport and altered residency time of cargoes in the Golgi can impair their functionality. To study the anterograde trafficking of specific protein cargoes, innovative molecular methods have been developed to synchronize the traffic of selected cargoes from the ER in live cells. These methods of synchronization now provide the ability to quantify the Golgi entry and exit kinetics of defined cargo. In this chapter, we describe a quantitative, accurate, and semiautomated protocol to image and quantify the anterograde trafficking of individual cargo traversing the Golgi. This protocol, using free software, is compatible with different synchronization techniques, and can be used for a range of applications, such as comparing the Golgi kinetics of (1) different cargoes, (2) wild-type cargo vs mutated cargo, (3) the same cargo under different Golgi conditions, and (4) cargoes in drug screening platforms. The method can also be applied to study the localization and transit of a cargo through different organelles other than the Golgi apparatus.

Interacting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down.

The small G protein Arl5b is localised on the trans-Golgi network (TGN) and regulates endosomes-to-TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane-bound Arl5b(Q70L)-GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP-Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN-localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.