Abstract
Membrane integration of a nascent opsin polypeptide was examined to determine whether insertion of proteins into the endoplasmic reticulum is dependent upon energy provided by ribonucleotide triphosphate hydrolysis. A discrete-sized nascent chain was obtained by in vitro translation of a mRNA which lacked a termination codon yet encoded the first 156 residues of bovine opsin. Ribosomes bearing the newly synthesized opsin chains were post-translationally incubated with canine pancreas microsomal membrane vesicles after addition of exogenous ribonucleotides or ribonucleotide analogues. Post-translational membrane integration and glycosylation of the 156-residue nascent polypeptide was found to require either the presence of guanosine triphosphate or a nonhydrolyzable GTP analogue. ATP did not promote post-translational integration of the nascent polypeptide. Although ribonucleotide hydrolysis was not obligatorily required for integration of opsin, we observed an increase in the proportion of glycosylated opsin chains in post-translational incubations that contained hydrolyzable ribonucleotide triphosphates. We conclude that a GTP-binding protein performs an essential role during integration of opsin into the endoplasmic reticulum.
Highlights
Quence to direct translocation of the amino-terminal lumenbinding protein performs an essential role during in- ally exposed domainof opsin and as a stop-transfesrequence tegration of opsin into the endoplasmic reticulum. to integrate the polypeptide in the membrane bilayer [9, 11]
The majority of currently available evidence indicates that both protein translocation and membrane protein integration occur primarily in a cotranslational fashion in mammalian cells
The low molecularweight opsin chain selected for this investigation should adequately represent the size class of polypeptidesthat are targeted to the endoplasmic reticulum in vivo
Summary
1mM emetine after 15min of translation inthe absence of membranes (c-h). The op-156 polysomes were separated from ribonucleotides by. Post-translational membrane integration experiments were of the nascent opsin chain (Fig. 3, lane e ). Glycosylation of performed by allowing translation to occur for 15 min prior op-156 was not observed in the presence of microsomal memto the addition of a protein synthesis elongation inhibitor branes plus 750 PM ATP (Fig. 3, lane d ) or 750 PM AMPPNP (cycloheximide or emetine). The nonhydrolyzable ATP analogue AMPPNP was included in the latter incubation as acompetitive inhibitor to insure that trace quantities of ATP could not account for the observed membrane integration of op-156.Aliquotsfrom the posttranslational incubations were removedat several time points and analyzed by digestionwith proteinase K (Fig. 5 andTable I) andby extraction with sodium carbonate (Table I). Theextent of glycosylation afterashort incubation with ribonucleotideswas modest (Fig. 5, lanes c and g, and quantitated in Table I), theglycosylatedopsin nascent chainswere
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