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.

Lipid droplets are sites of neutral lipid storage thought to be actively involved in lipid homeostasis. A popular model proposes that droplets are formed in the endoplasmic reticulum (ER) by a process that begins with the deposition of neutral lipids between the membrane bilayer. As the droplet grows, it becomes surrounded by a monolayer of phospholipid derived from the outer half of the ER membrane, which contains integral membrane proteins anchored by hydrophobic regions. This model predicts that for an integral droplet protein inserted into the outer half of the ER membrane to reach the forming droplet, it must migrate in the plane of the membrane to sites of lipid accumulation. Here, we report the results of experiments that directly test this hypothesis. Using two integral droplet proteins that contain unique hydrophobic targeting sequences (AAM-B and UBXD8), we present evidence that both proteins migrate from their site of insertion in the ER to droplets that are forming in response to fatty acid supplementation. Migration to droplets occurs even when further protein synthesis is inhibited or dominant-negative Sar1 blocks transport to the Golgi complex. Surprisingly, when droplets are induced to disappear from the cell, both proteins return to the ER as the level of neutral lipid declines. These data suggest that integral droplet proteins form from and regress to the ER as part of a cyclic process that does not involve traffic through the secretory pathway.

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that reside as inactive precursors in the endoplasmic reticulum (ER) membrane. After sterol depletion, the proteins are transported to the Golgi apparatus, where they are cleaved by site-1 protease (S1P). Cleavage releases the active transcription factors, which then enter the nucleus to induce genes that regulate cellular levels of cholesterol and phospholipids. This regulation depends on the spatial separation of the Golgi and the ER, as mixing of the compartments induces unregulated activation of SREBPs. Here, we show that S1P is localized to the Golgi, but cycles continuously through the ER and becomes trapped when ER exit is inhibited. During mitosis, S1P is associated with mitotic Golgi clusters, which remain distinct from the ER. In mitotic cells, S1P is active, but SREBP is not cleaved as S1P and SREBP reside in different compartments. Together, these results indicate that the spatial separation of the Golgi and the ER is maintained during mitosis, which is essential to protect the S1P substrate SREBP from unregulated activation during mitosis.