Several secretory proteins, including apolipoprotein B, have been shown to undergo degradation by proteasomes. We found that the rapid degradation of nascent apolipoprotein B in HepG2 cells was diminished but not abolished by the addition of any of three different inhibitors of proteasomes. Ubiquitin is conjugated to apolipoprotein B that is not assembled with sufficient lipids either during or soon after synthesis. In addition, we found that apolipoprotein B that has entered the endoplasmic reticulum sufficiently to become glycosylated can be degraded by proteasomes. Furthermore, we detected ubiquitin-apolipoprotein B that is associated with the Sec61 complex, the major constituent of the translocational channel. Treatment of cells with monomethylethanolamine or dithiothreitol decreased the translocation of apolipoprotein B and increased the proportion of ubiquitin-conjugated molecules associated with Sec61. Conversely, treatment of cells with oleic acid, which increased the proportion of translocated apolipoprotein B, decreased the amount of ubiquitin-apolipoprotein B in the Sec61 complex. Finally, we found that inhibition of the interaction between calnexin and apolipoprotein B decreases the translocation of apolipoprotein B, increases the ubiquitin-apolipoprotein B in the Sec61 complex, and increases the proteasomal degradation of glycosylated apolipoprotein B. Thus, ubiquitin can be attached to unassembled apolipoprotein B in the Sec61 complex, and this process is affected by factors including calnexin that alter the translocation of apolipoprotein B. Apolipoprotein B (apoB)1 is the large protein (Ͼ500 kDa) assembled with cholesterol and triglycerides into lipoprotein particles in hepatic and intestinal cells (1-4). Full-length apoB100 is secreted on very low density lipoproteins from hepatic cells, and apoB serves as a ligand on low density lipoproteins for the low density lipoprotein receptor. Increased levels of apoB and low density lipoprotein-associated cholesterol in human plasma correlate with increased risks of coronary artery disease.In HepG2 cells, a large amount of newly synthesized apoB is degraded (5-8). Intracellular disposal appears to be the principal means of regulating the secretion of apoB. Since an inhibitor of calpains and cysteine proteases, N-acetyl-leucylleucyl-norleucinal (ALLN), can protect apoB from degradation, an uncharacterized cysteine protease was proposed to be responsible for the apoB degradation (6, 7). ALLN also acts on proteasomes (9), and other chemicals that more specifically inhibit proteasomes also decrease the degradation of apoB (10 -12). In addition, ubiquitin has been found on apoB (10, 11). Several proteins that enter the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes (13-19). Recently, it has been shown that most if not all of these proteins undergo retrograde transport from the lumen of the ER back into the cytosol through a protein-conducting channel containing the Sec61 complex (20 -22). The Sec61 complex, which consists of ␣, ␤, and ␥ subu...

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, F., Le Cahérec, F., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J.F., Gouranton, J. & Thomas, D. (1995) J. Biol. Chem. 270, 17414-17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin 1, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert, J.F. (1996) J. Cell Sci. 109, 1285-1295]. In the present work, we cloned a full-length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26-kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein gamma, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15-fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrhenius activation energy of 11.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aquaporin 1. In the Xenopus oocyte system, Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions.

BackgroundOilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds. This weak N use efficiency is associated with a low foliar N remobilization, leading to a significant return of N to the soil and a risk of pollution. Contrary to what is observed during senescence in the vegetative stages, N remobilization from stems and leaves is considered efficient during monocarpic senescence. However, the contribution of stems towards N management and the cellular mechanisms involved in foliar remobilization remain largely unknown. To reach this goal, the N fluxes at the whole plant level from bolting to mature seeds and the processes involved in leaf N remobilization and proteolysis were investigated in two contrasting genotypes (Aviso and Oase) cultivated under ample or restricted nitrate supply.ResultsDuring seed filling in both N conditions, Oase efficiently allocated the N from uptake to seeds while Aviso favoured a better N remobilization from stems and leaves towards seeds. Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling. Under N limitation, Aviso had a better N remobilization from leaves to stems before the onset of seed filling. Afterwards, the higher N remobilization from stems and leaves of Aviso led to a higher final N amount in seeds. This high leaf N remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. By using an original method based on the determination of Rubisco degradation in the presence of inhibitors of proteases, efficient proteolysis associated with cysteine proteases and proteasome activities was identified as the mechanism of N remobilization.ConclusionThe results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms. In addition, the stem may act as transient storage organs in the case of an asynchronism between leaf N remobilization and N demand for seed filling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0437-1) contains supplementary material, which is available to authorized users.