Selective Release of Content From Microsomal Vesicles Without Membrane Disassembly

Abstract

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER) . More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons . The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation . A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content . Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER . Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and crossreacted with antiserum against rat serum . Almost all microsomal glycoproteins were at least partly released with the microsomal content .Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [ 3 H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER . On the other hand, after labeling for 30 min with [ 3 H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes . This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae .The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [ 3 H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue,

In vitro synthesis of the Ca 2+ transport ATPase by ribosomes bound to sarcoplasmic reticulum membranes

“…Although peripheral membrane proteins are removed from membranes by chelation of divalent cations or incubation in media of high ionic strength (28,29), the newly synthesized ATPase remained associated with the vesicles after ribosomes were detached by treatment with EDTA in a medium containing 0.5 M KC1. Furthermore, a major fraction of the in vitro synthesized ATPase was not released from microsomes by treatments that open microsomal vesicles and release their lumen contents (24,25). Only at deoxycholate concentrations that dissolved the membranes was a substantial fraction of the labeled ATPase released.…”

“…Subfractionation by differential centrifugation (1 hr at 45,000 X g) showed that the lower deoxycholate concentration used (0.1 mg/mg of microsomal protein in 0.3 ml containing 2.11 X 105 cpm) released 40% of the total microsomal-radioactivity, but labeled ATPase represented only 0.29% of the total radioactive protein in the released fraction. The bulk of the newly synthesized ATPase (0.98% of the radioactivity) was only released at a higher deoxycholate concentration (0.5 mg/mg of protein in 0.3 ml), which is known to cause extensive membrane dissolution (24,25). The major labeled product present in immunoprecipitates obtained from in vitro translation mixtures containing bound polysomes or rough microsomes had an electrophoretic mobility similar to that of the mature ATPase (Fig.…”

“…However, immunoprecipitation showed that the ribosome-stripped vesicles, which were recovered by centrifugation (2 hr at 100,000 X g) and dissolved with detergent, contained approximately 80% of the in vitro synthesized ATPase (1.12% of the total radioactivity). The stripped rough microsomal membranes were also treated with deoxycholate (30 min at 4°C) at concentrations that either release the microsomal content or dissolve the microsomal membranes (24,25). Subfractionation by differential centrifugation (1 hr at 45,000 X g) showed that the lower deoxycholate concentration used (0.1 mg/mg of microsomal protein in 0.3 ml containing 2.11 X 105 cpm) released 40% of the total microsomal-radioactivity, but labeled ATPase represented only 0.29% of the total radioactive protein in the released fraction.…”

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

“…Recently, studies performed in three granular systems other than the rabbit parotid--the guinea pig exocrine pancreas, the rat liver, and the bovine adrenal medulla--have given indication that removal of polypeptides known not to be membrane proteins to limiting levels of detectability has not been attained (47,37,26,68,70). In these studies the contaminating proteins are again secretory proteins identified by enzymatic activity, by comparative gel electrophoresis with soluble extracts of the fractions under investigation, or by a characteristic high rate of incorporation of radioactively tagged amino acid during pulse labeling.…”

“…In the case of rough microsomes isolated from rat liver, treatment with low concentrations of sodium deoxycholate, shown to create discontinuities in the microsomal membranes (35), promotes only partial extraction of short term-labeled (30-rain postpulse) polypeptides identified as protein destined for export from the liver (37). Thus both these studies suggest that adsorption to either the cytoplasmic or cisternal surfaces of membranebounded compartments as a consequence of cell or cellular compartment disruption in nonphysiologic media is both substantial and difficult to overcome by a variety of extractants.…”