Abstract: COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γâ-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.
Abstract: The p24 proteins function in early secretory pathway transport processes, but their exact role is unclear. In physiologically activated Xenopus melanotrope cells, a representative of each p24 subfamily (p24α(3), -β(1), -γ(3), -δ(2)) is upregulated coordinately with the major melanotrope cargo, proopiomelanocortin (POMC), whereas two other p24s (p24γ(2) and -δ(1)) are also expressed, but not coordinately with POMC. Using melanotrope-specific transgene expression, we here find that the roles of both p24γ(2) and p24δ(1) in the transport, glycosylation, sulphation and cleavage of POMC are different from those of their upregulated subfamily relatives (p24γ(3) and p24δ(2), respectively). Thus, even p24 proteins from the same subfamily have distinct functions in secretory cargo biosynthesis.
Abstract: COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.
Abstract: SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.
Abstract: The p24 protein family plays an important but unclear role at the ER (endoplasmic reticulum)-Golgi interface. A p24 member from each subfamily (p24alpha(3), beta(1), gamma(3) and delta(2)) is upregulated with the prohormone POMC (pro-opiomelanocortin) when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated. Here we explored the role of p24 by generating and analysing Xenopus with melanotrope cell-specific transgene expression of p24beta(1) or p24gamma(3), two of the p24 proteins coexpressed with POMC, and compared the results with those previously reported for the two other coexpressed p24s (p24alpha(3) and p24delta(2)).
Abstract: The p24 family is thought to be somehow involved in endoplasmic reticulum-to-Golgi protein transport, and its members are major constituents of transport vesicles and bind to the vesicle coat protein complexes COPI and COPII. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). To investigate the role of the COP-binding motifs of p24 proteins in POMC biosynthesis, we here generated and analysed Xenopus with stable, melanotrope cell-specific transgene expression of p24delta(2)-GFP mutated in its COPI- or COPII-binding motif. In contrast to what has been found previously for wild-type (wt) p24delta(2)-GFP, the p24delta(2) mutations prevented the Golgi localisation of the transgene products and caused a reduced rate of POMC cleavage, but did not lead to a reduction of the endogenous p24 proteins nor to aberrations in POMC glycosylation and sulphation. We conclude that p24delta(2) requires the presence of the COPI- and COPII-binding sites to allow proper POMC processing. Thus, the p24 proteins fulfil their role in secretory protein biosynthesis via COPI- or COPII-coated transport vesicles.
Abstract: The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type-I transmembrane proteins of approximately 24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER-Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.
Abstract: In black-background-adapted Xenopus laevis, the intermediate pituitary melanotrope cells are hyperactive, producing large amounts of their major secretory cargo proopiomelanocortin (POMC, representing ~80% of all newly synthesised proteins), whereas in white-adapted frogs these cells are only basally active. Here we explored in the hyperactive and basally active melanotrope cells the capacity for posttranslational POMC processing events in the secretory pathway.
Abstract: The members of the p24 protein family have an important but unclear role in transport processes in the early secretory pathway. The p24 family consists of four subfamilies (alpha, beta, gamma, and delta), whereby the exact composition of the family varies among species. Despite more than 15 years of p24 research, the vertebrate p24 family is still surprisingly ill characterized. Here, we describe the human, mouse, Xenopus, and zebrafish orthologues of 10 p24 family members and a new member that we term p24gamma(5). Of these eleven p24 family members, nine are conserved throughout the vertebrate lineage, whereas two (p24gamma(4) and p24delta(2)) occur in some but not all vertebrates. We further show that all p24 proteins are widely expressed in mouse, except for p24alpha(1) and p24gamma(5) that display restricted expression patterns. Thus, we present for the first time a comprehensive overview of the phylogeny and expression of the vertebrate p24 protein family.
Abstract: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC).
