The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.

The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks.Supplementary material available online at