Directed cell migration requires the orientation of the Golgi and centrosome toward the leading edge. We show that stimulation of interphase cells with the mitogens epidermal growth factor or lysophosphatidic acid activates the extracellular signal–regulated kinase (ERK), which phosphorylates the Golgi structural protein GRASP65 at serine 277. Expression of a GRASP65 Ser277 to alanine mutant or a GRASP65 1–201 truncation mutant, neither of which can be phosphorylated by ERK, prevents Golgi orientation to the leading edge in a wound assay. We show that phosphorylation of GRASP65 with recombinant ERK leads to the loss of GRASP65 oligomerization and causes Golgi cisternal unstacking. Furthermore, preventing Golgi polarization by expressing mutated GRASP65 inhibits centrosome orientation, which is rescued upon disassembly of the Golgi structure by brefeldin A. We conclude that Golgi remodeling, mediated by phosphorylation of GRASP65 by ERK, is critical for the establishment of cell polarity in migrating cells.

In vitro studies have suggested that Golgi stack formation involves two homologous peripheral Golgi proteins, GRASP65 and GRASP55, which localize to the cis and medial-trans cisternae, respectively. However, no mechanism has been provided on how these two GRASP proteins work together to stack Golgi cisternae. Here, we show that depletion of either GRASP55 or GRASP65 by siRNA reduces the number of cisternae per Golgi stack, whereas simultaneous knockdown of both GRASP proteins leads to disassembly of the entire stack. GRASP55 stacks Golgi membranes by forming oligomers through its N-terminal GRASP domain. This process is regulated by phosphorylation within the C-terminal serine/proline-rich domain. Expression of nonphosphorylatable GRASP55 mutants enhances Golgi stacking in interphase cells and inhibits Golgi disassembly during mitosis. These results demonstrate that GRASP55 and GRASP65 stack mammalian Golgi cisternae via a common mechanism.

Significance: In Alzheimer’s disease (AD), formation of the Aβ aggregates occurs by the cleavage of the amyloid precursor protein (APP) during its trafficking inside the nerve cells. The Golgi apparatus plays a critical role in APP trafficking; fragmentation of the normally highly ordered Golgi structure occurs in nerve cells of AD patients. Here we report that Aβ accumulation triggers Golgi fragmentation by activating cyclin-dependent kinase-5 (cdk5), which phosphorylates Golgi structural proteins such as GRASP65. Rescue of Golgi structure by inhibiting cdk5 or by expressing nonphosphorylatable GRASP65 mutants reduced Aβ secretion. Our study provides a molecular mechanism for Golgi fragmentation and its effects on APP trafficking and processing, suggesting Golgi as a potential drug target for AD treatment.

The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum, processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network. GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking and unconventional secretion. Previously, we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion or endoplasmic reticulum stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the trans-Golgi network.

Cell‐free assays that mimic the disassembly and reassembly cycle of the Golgi apparatus during mitosis implicated GRASP65 as a mitotically regulated stacking factor. We now present evidence that GRASP65 is directly involved in stacking Golgi cisternae. GRASP65 is the major phosphorylation target in rat liver Golgi membranes of two mitotic kinases, cdc2–cyclin B and polo‐like kinases, which alone will unstack Golgi membranes, generating single cisternae. Mitotic cells microinjected with antibodies to GRASP65 fail to form proper Golgi stacks after cell division. Beads coated with GRASP65 homodimers form extensive aggregates consistent with the formation of trans oligomers. These can be disaggregated using purified cdc2–cyclin B1 and polo‐like kinases, and re‐aggregated after dephosphorylation of GRASP65. Together, these data demonstrate that GRASP65 has the properties required to bind surfaces together in a mitotically regulated manner.