BML-265 and Tyrphostin AG1478 Disperse the Golgi Apparatus and Abolish Protein Transport in Human Cells.

Gaelle Boncompain,Nelly Gareil,Thouis R Jones,Sarah Tessier,Guido Kroemer,Elaine Del Nery,Aurianne Lescure,Oliver Kepp,Franck Perez

doi:10.3389/fcell.2019.00232

Abstract

The steady-state localization of Golgi-resident glycosylation enzymes in the Golgi apparatus depends on a balance between anterograde and retrograde transport. Using the Retention Using Selective Hooks (RUSH) assay and high-content screening, we identified small molecules that perturb the localization of Mannosidase II (ManII) used as a model cargo for Golgi resident enzymes. In particular, we found that two compounds known as EGFR tyrosine kinase inhibitors, namely BML-265 and Tyrphostin AG1478 disrupt Golgi integrity and abolish secretory protein transport of diverse cargos, thus inducing brefeldin A-like effects. Interestingly, BML-265 and Tyrphostin AG1478 affect Golgi integrity and transport in human cells but not in rodent cells. The effects of BML-265 are reversible since Golgi integrity and protein transport are quickly restored upon washout of the compounds. BML-265 and Tyrphostin AG1478 do not lead to endosomal tubulation suggesting that, contrary to brefeldin A, they do not target the trans-Golgi ARF GEF BIG1 and BIG2. They quickly induce COPI dissociation from Golgi membranes suggesting that, in addition to EGFR kinase, the cis-Golgi ARF GEF GBF1 might also be a target of these molecules. Accordingly, overexpression of GBF1 prevents the effects of BML-265 and Tyrphostin AG1478 on Golgi integrity.

Highlights

The Golgi apparatus lies at the center of the secretory pathway of eukaryotic cells
Mannosidase II (ManII)-Streptavidin Binding Peptide (SBP)-EGFP is retained in the ER in the absence of biotin and is transported to the Golgi apparatus after incubation with biotin (Figure 1B)
AMF-26/M-COPA, Golgicide A, Exo2, LG-186 and Tyrphostin AG1478 were identified as molecules displaying effects similar to brefeldin A (BFA), but being more specific because they target the ADP-ribosylation factors (ARF) guanine nucleotide exchange factors (GEF) Golgi brefeldin A-resistant factor 1 (GBF1) (Pan et al, 2008; Spooner et al, 2008; Saenz et al, 2009; Boal et al, 2010; Ohashi et al, 2012)

Summary

Introduction

The Golgi apparatus lies at the center of the secretory pathway of eukaryotic cells. This organelle receives neo-synthesized secretory proteins from the ER and sorts them to their destination compartments, such as the endosomes or the plasma membrane (Boncompain and Weigel, 2018). Defects in the function and/or localization of Golgi associated proteins regulating trafficking and especially of glycosylation enzymes could lead to diverse diseases (Zappa et al, 2018). At the cis-Golgi, the Golgi brefeldin A-resistant factor 1 (GBF1) plays a pivotal role in regulating organelle structure and vesicle trafficking by catalyzing the activation of ARF1 leading to COPI assembly and recruitment to Golgi membranes (Presley et al, 2002)

Methods

Results

Conclusion