The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Abstract

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein . N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membraneattached polysomes and not into proteins from free polysomes . Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes .Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae . Albumin was mostly recovered (98%0 ) in the cisternae, with negligible amounts in the membranes . To determine whether the radioactive sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the "C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins . The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae .

Selective Release of Content From Microsomal Vesicles Without Membrane Disassembly

“…Smooth microsomes labeled for short periods of time showed a 10-fold higher incorporation of [ 3 H]glucosamine than RM . This observation is also consistent with the sequence of glycosidation steps which is known to involve enzymes in rough and smooth endoplasmic reticulum membranes after synthesis, and release of recently synthesized proteins from membrane-bound ribosomes (Molnar et al ., 1965 ;Schachter et al ., 1970 ;Redman and Cherian, 1972 Kreibich and Sabatini, 1971) .…”

“…In rat liver the bulk of the protein-synthesizing activity of the rough endoplasmic reticulum is devoted to the manufacture of serum proteins (Peters et al ., 1971 ;Redman and Cherian, 1972), several of which are known to be glycoproteins . It was therefore of interest to compare the electrophoretic pattern of serum proteins and glycoproteins with those of proteins and glycoproteins in microsomal subfractions prepared using different DOC concentrations .…”

“…It was previously shown that after 30 min of [ 3 H]glucosamine administration, the smooth microsomal fraction contains per milligram of protein almost 10 times more glycoprotein radioactivity than rough microsomes ; see also Schachter et al ., 1970 ;Fleischer and Fleischer, 1970 ;Wagner and Cynkin, 1971 ;Sturgess et al ., 1972 ;Redman and Cherian, 1972) . In spite of this difference in the level of radioactivity, similar sets of labeled glycoproteins were found by acrylamide gel electrophoresis in RM, SM, and their subfractions (Fig .…”

“…In liver cells, secretory proteins destined for the bloodstream are thought to be manufactured in membrane-bound polysomes and to be directly discharged into the ER cisternae (Palade and Siekevitz, 1956 ;Campbell et al ., 1960 ;Peters, 1962 a,b ; Redman and Sabatini, 1966 ;Redman, 1969 ;Ganoza and Williams, 1969), where they may be modified by enzymes bound to the limiting membranes (Molnar et al ., 1965 ;DeLorenzo et al ., 1966 ;Wagner and Cynkin, 1971 ;Redman and Cherian, 1972), before being transferred to the Golgi apparatus (Glaumann, 1970 ;Glaumann and Ericsson, 1970;Schachter et al ., 1970 ;Peters et al ., 1972) . The intracisternal route provided by the ER may also be utilized by proteins retained intracellularly which are diverted from the secretory pathway into other membrane-bound compartments, such as lysosomes (de Duve and Wattiaux, 1966 ;Goldstone and Koenig, 1972), or peroxisomes (Higashi and Peters, 1963 a,b ; Kashiwagi et al ., 1971 ; but see Lazarow andde Duve, 1973) .…”

The synthesis and secretion of albumin

“…After intravenous injection of [3 H] leucine (LeBouton, 1968), the kinetics of labeling of intrahepatic albumin and plasma albumin showed the relationship postulated for a precursor and its product by Zilversmit et al in 1943. The kinetics of labeling of albumin in various subcellular compartments presented a more complicated pattern (Peters, 1962b;Peters et al, 1971;Glaumann, 1970;Glaumann and Ericsson, 1970;Redman and Cherian, 1972;Jamieson and Ashton, 1973). Maximal labeling of albumin occurred first in the smooth, then in the rough endoplasmic membranes, and finally in the Golgi apparatus.…”