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A Ceriotti

Publications and source records attributed to A Ceriotti.

26 records · Page 2Linked to original sources
PubMed1992

Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum.

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.

Amino Acid Sequence↗
PubMed1991

Expression of the wild-type and mutated vacuolar storage protein phaseolin in Xenopus oocytes reveals relationships between assembly and intracellular transport.

The role played by subunit assembly in the intracellular transport of the bean storage protein phaseolin, a soluble trimeric glycoprotein, was investigated using Xenopus oocytes injected with RNA. We show that phaseolin assembly is dependent upon the level of synthesis of the protein and is required for intracellular transport out of the endoplasmic reticulum. We also show that a fraction of the assembled phaseolin is permanently retained in a post-endoplasmic reticulum compartment. Deletion of the C-terminal alpha-helical domain fully prevents in vivo assembly but not endoplasmic reticulum retention. This indicates that this domain is necessary for trimerization but not for interactions of unassembled subunits with endoplasmic reticulum components. The truncated phaseolin has high in vivo stability. The potential implications of these findings on the possibility to improve the nutritional value of phaseolin through genetic engineering are discussed.

Animals↗
PubMed1991

A Saporin-6 cDNA containing a precursor sequence coding for a carboxyl-terminal extension.

Saporin-6 is a single-chain ribosome inactivating protein (RIP) from the seeds and the leaves of Saponaria officinalis (Caryophyllaceae). Here we have identified the COOH-terminal end of mature Saporin-6 and, by cDNA sequencing, the predicted carboxyl-terminal sequence of a leaf Saporin-6 primary translation product. Our data indicate that the characterized cDNA codes for a precursor containing a 22 amino acid carboxyl-terminal extension, not present in mature Saporin-6, that shows similarity to carboxyl-terminal propeptides of vacuolar proteins, suggesting that it may be involved in protein trafficking.

Amino Acid Sequence↗
PubMed1990

Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes.

We have previously shown that influenza haemagglutinin (HA) acquires Endo H resistance en route to the cell surface after microinjection of its mRNA into Xenopus oocytes (Ceriotti, A. and A. Colman. 1989. J. Cell Biol. 109:1439-1444.) In this paper we use the injection of varying amounts of mRNA (0.05-5 ng/oocyte) to effect a 30-fold change in HA protein synthesis within the oocyte. Using the Endo H assay as an indicator of protein movement from the ER to the medial Golgi we find that this movement is reduced, sometimes dramatically, when intracellular HA levels fall. This reduction in movement is closely correlated with a decreased rate of trimer formation as assessed both by trypsin resistance and sedimentation analysis, leading us to conclude that trimer formation is not only, as has been shown before essential for ER-Golgi complex movement, but is the major rate limiting step in this movement. Interestingly at least 50% of unassembled HA monomers that accumulate after low HA synthesis can be rescued into trimers over 24 h later, after a second injection of concentrated HA mRNA. In contrast when we repeated this experiment with another membrane protein, the human low density lipoprotein, or with murine secretory immunoglobulin we found that the rate of movement was insensitive to the protein concentration. This latter result seemed surprising since earlier work had shown that unassembled IgG heavy chains (like monomeric HA) remain in the oocyte ER; however in these present experiments we have been unable to detect any unassembled heavy chains even at the lowest expression levels, indicating that tetramerization of Ig is much faster than trimerization of HA.

Animals↗
PubMed1989

Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes.

We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium. In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes. Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion. In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period. We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation. This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected.

Animals↗
PubMed1988

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.

Animals↗
PubMed1984

Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons.

Phytohemagglutinin (PHA) is a family of tetrameric isolectins which accumulate in the protein bodies of developing Phaseolus vulgaris cotyledons. Each tetramer contains erythroagglutinating (E) or lymphocyte-mitogenic (L) subunits, or a combination of both. The subunits have Mr around 33000, E being slightly larger than L. Phytohemagglutinin is a glycoprotein, and its carbohydrate moiety contains N-acetylglucosamine, mannose, fucose and xylose, indicating that this protein has complex oligosaccharide sidechains. Several steps in the biosynthesis and in the cotranslational and post-translational processing of the glycopolypeptides of PHA have been identified. The polypeptides of PHA are synthesized by polysomes attached to the endoplasmic reticulum. The glycosylation of the polypeptides is a cotranslational process, in which each PHA polypeptide usually acquires two oligosaccharide sidechains. The oligosaccharides of PHA isolated from the endoplasmic reticulum are susceptible to digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H indicating that they are of the high-mannose type. In the presence of tunicamycin two unglycosylated polypeptides of PHA are synthesized, indicating that the differences in Mr between the E and L subunits of PHA are not due to differences in glycosylation alone. Transport of PHA to the protein bodies is mediated by the Golgi apparatus where at least part of the oligosaccharide chains of PHA are modified [ Chrispeels , M. J. (1983) Planta ( Berl .) 157, 454-461, and 158, 140-151]. The modified oligosaccharide chains of PHA are then gradually trimmed to a smaller size when the protein is already in the protein bodies. This processing results in an increase in the mobility of the PHA subunits in denaturing polyacrylamide gels.

Endoplasmic Reticulum↗