S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.

Exocytosis is the process whereby intracellular fluid-filled vesicles fuse with the plasma membrane, incorporating vesicle proteins and lipids into the plasma membrane and releasing vesicle contents into the extracellular milieu. Exocytosis can occur constitutively or can be tightly regulated, for example, neurotransmitter release from nerve endings. The last two decades have witnessed the identification of a vast array of proteins and protein complexes essential for exocytosis. SNARE proteins fill the spotlight as probable mediators of membrane fusion, whereas proteins such as munc18/nsec1, NSF and SNAPs function as essential SNARE regulators. A central question that remains unanswered is how exocytic proteins and protein complexes are spatially regulated. Recent studies suggest that lipid rafts, cholesterol and sphingolipid-rich microdomains, enriched in the plasma membrane, play an essential role in regulated exocytosis pathways. The association of SNAREs with lipid rafts acts to concentrate these proteins at defined sites of the plasma membrane. Furthermore, cholesterol depletion inhibits regulated exocytosis, suggesting that lipid raft domains play a key role in the regulation of exocytosis. This review examines the role of lipid rafts in regulated exocytosis, from a passive role as spatial coordinator of exocytic proteins to a direct role in the membrane fusion reaction. The intracellular transport of proteins and lipids relies to a large extent on their sorting into specific vesicle populations, the directional movement of the vesicles through the cell, and the subsequent fusion of the vesicles with specific cellular compartments. The fusion of vesicles with the plasma membrane (PM) occurs in a process called exocytosis; this membrane fusion event mediates the targeting of proteins and lipids to the PM and the secretion of molecules from the cell. Exocytosis can occur constitutively or can be tightly regulated.Constitutive exocytosis events include the fusion of vesicles derived from the trans-Golgi network (TGN) with the PM, which is essential for the insertion of newly synthesized proteins and lipids into the PM. Polarized cells have developed specialized mechanisms for the targeting of these TGN-derived vesicles to specific regions of the PM, for example, apical vs. basolateral membrane in polarized epithelial cells (1). In addition, proteins that are constitutively recycled through the endosomal system, such as the transferrin receptor, are transported to the cell surface via the fusion of endosomal vesicles with the PM. These constitutive pathways operate in all cells. In addition, a number of cell types undergo a more specialized form of exocytosis known as regulated exocytosis. Exocytosis of regulated secretory vesicles only occurs upon receipt of a specific stimulus, such as exocytosis of synaptic vesicles in nerve cells. In the majority of cases, regulated exocytosis is stimulated by a local and transient increase in calcium levels (2).Exocytosis can involve the full fusion of a vesicle with the ...

Background PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na+-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.Methodology/Principal FindingsTo determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.Conclusion/SignificanceThis work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.