Our results show that COPII coat recruitment by cargo receptors is not constitutive but instead is actively regulated by binding of mature ligands. Therefore, we reveal a novel functional link between luminal cargo maturation and COPII vesicle budding, providing a mechanism to adjust specialized COPII vesicle production to the amount and quality of their luminal cargos that are ready for ER exit. This helps to understand how the ER export machinery adapts to different needs for luminal cargo secretion.

Wine production is an ancient human activity that generates several by-products, which include some constituents known for their potential in health care and for their role in the food or cosmetic industries. Any variety of grape (Vitis vinifera L.) contains nutrients and bioactive compounds available from their juice or solid parts. Grape seed extract has demonstrated many activities in disease prevention, such as antioxidant effects, which make it a potential source of nutraceuticals. Grape seed is a remarkable winery industry by-product due to the bioactivity of its constituents. Methods for recovery of oil from grape seeds have evolved to improve both the quantity and quality of the yield. Both the lipophilic and hydrophilic chemicals present in the oil of V. vinifera L. make this wine by-product a source of natural nutraceuticals. Food and non-food industries are becoming novel targets of oil obtained from grape seeds given its various properties. This review focuses on the advantages of grape seed oil intake in our diet regarding its chemical composition in industries not related to wine production and the economic and environmental impact of oil production.

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.

We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied in vitro using vesicular stomatitis virusinfected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN.Transport of newly synthesized proteins along the exocytic pathway is mediated by small vesicular carriers (1, 2). A set of proteins collectively known as the transport machinery is responsible for the formation and consumption of vesicles. These include receptor proteins that sort and concentrate cargo molecules at the budding site, coat proteins that catalyze budding from the donor compartment, and proteins responsible for targeting and fusion with the acceptor membrane (1). Recent evidences indicate that the transport machinery is regulated by specific phosphorylating activities that in this way modulate intracellular traffic. Thus, phosphatidylinositol-3 kinase has been shown to be required for transfer of hydrolases from the Golgi to either the yeast vacuole (3) or the mammalian lysosomes (4, 5). Release of secretory vesicles from the trans-Golgi network (TGN) of endocrine cells has also been shown to be regulated by tyrosine phosphorylation (6). Both protein kinase A (PKA) and protein kinase C (PKC) activities have been reported to control particular vesiclemediated transport steps such as constitutive (7-11) and regulated exocytosis (12-15) and transcytosis (16,17). Whereas the effects derived from the activation͞inhibition of these different kinases on transport are well documented little information is available about their mechanism of action. PKC activity has been described to stimulate association of ADP ribosylation factor and coatomer proteins to Golgi membranes (18) but recent data indicate that its phosphorylating activity is not required for vesicle budding from the TGN (19). Therefore, it is uncertain at present in which way protein kinases control transport activities. We have recently described that PKA differentially regulates constitutive protein transport along the exocytic pathway with transport from the TGN to the cell surface being strictly dependent on PKA ...

The small GTP-binding protein ADP-ribosylation factor 1 (ARF1) is an essential component of the molecular machinery that catalyzes the formation of membranebound transport intermediates. By using an in vitro assay that reproduces recruitment of cytosolic proteins onto purified, high salt-washed Golgi membranes, we have analyzed the role of cAMP-dependent protein kinase A (PKA) on ARF1 incorporation. Addition to this assay of either pure catalytic subunits of PKA (C-PKA) or cAMP increased ARF1 binding. By contrast, ARF1 association was inhibited following C-PKA inactivation with either PKA inhibitory peptide or RII␣ as well as after cytosol depletion of C-PKA. C-PKA also stimulated recruitment and activation of a recombinant form of human ARF1 in the absence of additional cytosolic components. The binding step could be dissociated from the activation reaction and found to be independent of guanine nucleotides and saturable. This step was stimulated by C-PKA in an ATP-dependent manner. Dephosphorylated Golgi membranes exhibited a decreased ability to recruit ARF1, and this effect was reverted by addition of C-PKA. Following an increase in the intracellular level of cAMP, ARF proteins redistributed from cytosol to the perinuclear Golgi region of intact cells. Collectively, the results show that PKA exerts a key regulatory role in the recruitment of ARF1 onto Golgi membranes. In contrast, PKA modulators did not affect recruitment of ␤-COP onto Golgi membranes containing prebound ARF1.