Background KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. Results We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. Conclusions These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.

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Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and β-tubulin. However, very little is known about the specific modifications found on the different tubulin isotypes in vivo and the role of these PTMs in MT transport and other cellular processes in vivo. In this study, we found that in Drosophila ovaries, glutamylation of α-tubulin isotypes occurred clearly on the C-terminal ends of αTub84B and αTub84D (αTub84B/D). In contrast, the ovarian α-tubulin, αTub67C, is not glutamylated. The C-terminal ends of αTub84B/D are glutamylated at several glutamyl sidechains in various combinations. Drosophila TTLL5 is required for the mono- and poly-glutamylation of ovarian αTub84B/D and with this for the proper localization of glutamylated microtubules. Similarly, the normal distribution of Kinesin-1 in the germline relies on TTLL5. Next, two Kinesin-1 dependent processes, the precise localization of Staufen and the fast, bidirectional ooplasmic streaming, depend on TTLL5, too, suggesting a causative pathway. In the nervous system, a mutation of TTLL5 that inactivates its enzymatic activity decreases the pausing of anterograde axonal transport of mitochondria. Our results demonstrate in vivo roles of TTLL5 in differential glutamylation of α-tubulins and point to the in vivo importance of α-tubulin glutamylation for cellular functions involving microtubule transport.

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Microtubules (MTs) are built from α-/β-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and β-tubulin. However, very little is known about the specific modifications found on the different tubulin isoforms in vivo and the role of these PTMs in cargo transport along MTs in vivo. In this study, we found that in Drosophila, glutamylation of the α-tubulin isoforms occurs specifically on the C-terminal ends of TBA1 and TBA3 in the ovaries. In contrast, the ovarian isoform TBA4 is not glutamylated. The C-terminal ends of TBA1 and TBA3 are glutamylated at several glutamyl side chains in various combinations. Drosophila TTLL5 is required for the mono- and polyglutamylation of ovarian TBA1 and 3. Furthermore, glutamylation of the α-tubulin is essential for the efficient localization of Staufen/osk mRNA and to give directionality to the fast ooplasmic streaming, two processes known to depend on kinesin-mediated processes during oogenesis. In the nervous system, the kinesin-dependent neuronal transport of mitochondria also depends on TTLL5. Additionally, α-tubulin glutamylation affects the pausing of the transport of individual mitochondria in the axons. Our results demonstrate the in vivo role of TTLL5 in differential glutamylation of α-tubulin isoforms and point to the in vivo importance of α-tubulin glutamylation for kinesin-dependent processes.