Abstract
Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.
Highlights
The actin cytoskeleton is linked to trafficking in the early secretory pathway [3,4,5,6,7]
We have found that the actin-binding proteins mammalian Abp1 (mAbp1), drebrin, and cortactin are recruited to Golgi membranes upon activating ARF1 [5, 6, 17]
These results show that regulated binding properties of GST-drebrin and GST-mAbp1 are the same as those reported previously for the endogenous cytosolic proteins
Summary
Materials—Rat liver Golgi membranes and bovine brain cytosol were isolated as described previously [28]. The cDNA fragments encoding the different truncations and chimeras of mAbp and drebrin were obtained by PCR. The first PCR reactions utilized an appropriate chimeric oligonucleotide. The product from the first reaction was used as a primer for the second reaction to complete a cDNA encoding the desired chimeric protein. Chimeric, and full-length drebrin and mAbp as GST fusion proteins, they were cloned into the bacterial expression vector pGEX-4T-2. The expressed proteins were purified using glutathione-Sepharose as described previously [5, 6]. The encoding cDNAs were ligated into pEGFP-C1 or pEGFP-N1 plasmids for expression as GFP fusion proteins in Vero cells. Golgi Membrane Binding Assays—Golgi membrane binding and budding reactions were carried out as described previously [3, 5, 6, 29]. Immunofluorescence and confocal microscopy (Zeiss) were carried out as described previously [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
