N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles.The movement of proteins between membrane-bound compartments is carried out by transport vesicles (1). Elucidation of the molecular mechanisms involved in vesicle budding and fusion will require that the components of the transport machinery be isolated in functional form. Here, we report the purification to essential homogeneity of one such protein component. This protein, the N-ethylmaleimide-sensitive factor (NSF), is needed for biosynthetic transport between Golgi cisternae in cell-free systems (2, 3).Biosynthetic protein transport occurs in two distinct phases. First, a chain of nonselective "bulk-carrier" vesicles move proteins from the endoplasmic reticulum to the Golgi, from cisternae to cisternae across the Golgi stack, and into the trans-Golgi network (4). Then, in the trans-Golgi network, the proteins are separated according to their destinations (5-7). Much of the first part of this pathway has been reconstituted in a cell-free system that measures the transport of the vesicular stomatitis virus-encoded glycoprotein (VSV-G protein) between successive cisternae of the Golgi stack (8-13). Transport requires ATP as well as cytosol (high-speed supernatant), and appears to be mediated by non-clathrincoated vesicles that are the bulk carriers mentioned above (12,14).We have reported (2) that treatment with N-ethylmaleimide (NEM) under mild conditions (1 mM, 15 min, 0°C) selectively inactivated Golgi membranes in the cell-free transport assay. Transport could be restored to NEM-treated Golgi membranes by adding back the ATP extract of untreated Golgi membranes. This restorative factor was itself sensitive to NEM. The activity of NSF is stimulated by long chain fatty acyl-CoA (2, 3). NSF is required for transport at multiple levels of the Golgi stack (3). Here we report the purification of NSF from CHO cytosol prepared in the presence of ATP.MATERIALS AND METHODS Preparation of Golgi Membranes and NSF-Free Cytosol. Donor and acceptor membranes were prepared as described (8,15) The NSF fraction to be tested (up to 20 pl) was added last. After incubation at 370C for 1 hr, the VSV-G protein was immunoprecipitated at 40C for at least 6 hr as described (8). Assays of all fractions were performed in a predetermined linear range (0-0.2 mg/ml for crude ATPstabilized cytosol). Large-Scale Preparation of ATP-Containing Cytosol for NSF Purifications. A washed pellet of CHO cells (1 vol) was resuspended with 4 vol of swelling buffer containing 20 mM Pipes-KOH (pH 7.2), 10 mM MgCl2, 5 mM ATP, 5 mM dithio...
A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.
Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.
Abstract. Type 13 transforming growth factor (TGFI3) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGFI3 also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGFI3 for control of myogenic differentiation. In mouse C-2 myoblasts, TGFfl inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC3H1, was also inhibited by TGFI3 in a dose-dependent manner (IDs0 ~0.5 ng/ml). TGFI3 was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGFI3 in the culture media. Removal of TGFI3 led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGFfl are highly transient and require continuous occupancy of the TGFI~ receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGFI3 inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGFI3 is a potent regulator of myogenic differentiation and suggest that TGFI3 may play an important role in the control of tissue-specific gene expression during development.
Several proteins of viral and cellular origin are acylated with myristic acid early during their biogenesis. To investigate the possibility that myristylation occurred cotranslationally, the BC3H1 muscle cell line, which contains a broad array of myristylated proteins, was pulse-labeled with [3H]myristic acid. Nascent polypeptide chains covalently associated with transfer RNA were isolated subsequently by ion-exchange chromatography. [3H]Myristate was attached to nascent chains through an amide linkage and was identified by thin-layer chromatography after its release from nascent chains by acid methanolysis. Inhibition of cellular protein synthesis with puromycin resulted in cessation of [3H]myristate-labeling of nascent chains, in agreement with the dependence of this modification on protein synthesis in vivo. These data represent a direct demonstration that myristylation of proteins is a cotranslational modification.
Abstract. The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked marmose in the ER. The earliest detectable species, 11 (ti129 kD), under-
Abstract-Transcriptome-wide analysis of dynamically regulated progenitor cell populations has the potential to elucidate key aspects of cardiac development. The heart, as the first organ to develop in the mammal, is a technically challenging but clinically relevant target for study. To define the transcriptional program of the cardiac progenitor, we used a novel transgenic strategy and fluorescence-activated cell sorting to reliably label and isolate cardiac progenitors directly from mouse embryos. Pure populations of cardiac progenitor cells were isolated from the cardiac crescent and 2 subsequent stages of heart development: the linear heart tube and the looping heart. RNA was isolated from stage-specific cardiac progenitors and subjected to transcriptome analysis by oligonucleotide array hybridization. The cardiac transcriptional regulatory programs were compared with the molecular programs of age-matched noncardiac embryonic cells, embryonic stem cells, adult cardiomyocytes, and each other to identify sets of genes exhibiting differential expression in the cardiac progenitor cell population. These results define the transcriptional profile of mammalian cardiac progenitor cells and provide insight into the molecular regulation of the earliest periods of heart development.
The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.
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