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The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Delta, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Delta cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

Mutations in the FGD1 gene are responsible for the X-linked disorder known as faciogenital dysplasia (FGDY). FGD1 encodes a guanine nucleotide exchange factor that specifically activates the GTPase Cdc42. In turn, Cdc42 is an important regulator of membrane trafficking, although little is known about FGD1 involvement in this process. During development, FGD1 is highly expressed during bone growth and mineralization, and therefore a lack of the functional protein leads to a severe phenotype. Whether the secretion of proteins, which is a process essential for bone formation, is altered by mutations in FGD1 is of great interest. We initially show here that FGD1 is preferentially associated with the trans-Golgi network (TGN), suggesting its involvement in export of proteins from the Golgi. Indeed, expression of a dominant-negative FGD1 mutant and RNA interference of FGD1 both resulted in a reduction in post-Golgi transport of various cargoes (including bone-specific proteins in osteoblasts). Live-cell imaging reveals that formation of post-Golgi transport intermediates directed to the cell surface is inhibited in FGD1-deficient cells, apparently due to an impairment of TGN membrane extension along microtubules. These effects depend on FGD1 regulation of Cdc42 activation and its association with the Golgi membranes, and they may contribute to FGDY pathogenesis.

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p-14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling.