Antiribophorin antibodies inhibit the targeting to the ER membrane of ribosomes containing nascent secretory polypeptides.

Abstract

Abstract. Polyclonal antibodies directed against ribophorins I and II, two membrane glycoproteins characteristic of the rough endoplasmic reticulum, inhibit the cotranslational translocation of a secretory protein growth hormone into the lumen of dog pancreas or rat liver microsomes. As expected, sitespecific antibodies to epitopes located within the cytoplasmic domain of ribophorin I, but not antibodies to epitopes in the luminal domain of this protein, were effective in inhibiting translocation. Since monovalent Fab fragments were as inhibitory as intact IgG molecules, ribophorins must be closely associated with the translocation site and, therefore, are likely to function at some stage in the translocation process. In all cases, the antibodies that inhibited translocation also caused a significant reduction in total protein synthesis and treatments that neutralized their capacity to inhibit translocation also prevented their inhibitory effect on protein synthesis. This would be expected if the antibodies blocked the membrane-mediated relief of the SRP-induced arrest of polypeptide elongation. The antibodies were effective only when added before translocation was allowed to begin. In this case, they prevented the targeting of active ribosomes containing mRNA and nascent chains to the ER membrane. Thus, ribophorins must either directly participate in targeting or be so close to the targeting site that the antibodies sterically blocked this early phase of the translocation process.

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

“…5,6,46 Secondly, the well-characterized, genetically modified strains used to describe the bacterial ribosome-binding site 20,21 do not exist in Archaea. Thirdly, studies exploiting antibodies raised against translocon components to delineate the ribosome -translocon interaction 47,48 are not feasible with halophilic archaea such as H. volcanii, given that the molar concentrations of salt required for the study of biochemical and cellular processes in this species would interfere with antibody binding. Finally, until only recently, 39 no reliable protocol for large-scale preparation of archaeal IMVs was available.…”

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones

“…VL3-3M2 cells, as well as the BW5147 thymic lymphoma cell line (33), were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). The rabbit antibodies (Ab) used in this study were raised against the following immunogens: (i) anti-cal-N, fusion protein encompassing the N-terminal 374 aa of mouse calnexin (24); (ii) anti-cal-C, C-terminal 12 aa of mouse calnexin (34); (iii) anti-rI, aa 563-583 of rat ribophorin I (35); (iv) anti-rII, aa 1-22 of ribophorin II (35); (v) anti-SSR, aa 266-286 of signal sequence receptor ␣ subunit (36); and (vi) anti-CRT, recombinant human calreticulin (CRT) (Affinity BioReagents, Golden, CO). The following mAb were used: (i) anti-CD3 , 145-2C11 (37) and (ii) anti-KDEL (StressGen Biotechnologies, Victoria, BC).…”

Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.

“…To ascertain the relative presence of the various membrane sources in the fractions obtained (F1-F4), immunoblot analyses were performed with antibodies against organelle-specific markers: anti-a subunit of the Na+/K+-ATPase for PM (26), anti-ribophorin for endoplasmic reticulum (ER) (27), and anti-TGN 38/41 for Golgi membranes (28). A representative experiment performed with fractions from TSH(-) cells is shown in Fig.…”