No abstract

The enzyme phospholipase A2 (cPLA2α) is involved in the formation of intercisternal tubules that mediate transport of proteins within the Golgi complex.

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.

a b s t r a c tThe particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.

Previous studies have demonstrated that membrane tubulemediated transport events in biosynthetic and endocytic routes require phospholipase A 2 (PLA 2 ) activity. Here, we show that cytosolic phospholipase A 2 e (cPLA 2 e, also known as PLA2G4E) is targeted to the membrane compartments of the clathrinindependent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P 2 ] that are enriched in clathrin-independent endosomes. Ablation of cPLA 2 e suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA 2 e to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA 2 e S420A mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA 2 e is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA 2 e for this pathway supports a new hypothesis that different PLA 2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.