Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

Yoshida, Hisashi; Tondokoro, Naoki; Asano, Yoji; Mizusawa, Kiyoshi; Yamagishi, Ryohei; Horigome, Tsuneyoshi; Sugano, Hiroshi

doi:10.1042/bj2450811

Cited by 20 publications

(23 citation statements)

References 29 publications

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“…Rat liver RM were treated with 0.5 mM puromycin/0.5 M KCI as described [4,9]. The stripped microsomes possessed a number of binding sites, 65 nmol/g membrane proteins, as determined by the method of Yoshida et al [9].…”

Section: Inhibition Of Ribosome Binding By Antibodiesmentioning

confidence: 99%

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Anti‐(p34 protein) antibodies inhibit ribosome binding to and protein translocation across the rough microsomal membrane

Ichimura¹,

Shindo²,

Uda³

et al. 1993

FEBS Letters

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The p34 protein is a non-glycosylated, integral membrane protein characteristic of rough microsomes and is believed to play a role in the ribosome-membrane association. Here, antibodies directed against p34 were examined as to their inhibitory effect on ribosome binding to and protein translocation across the microsomal membrane. Preincubation of the stripped (ribosome-depleted) membrane with anti-p34 immunoglobulins (IgGs) or their Fab fragments led to more than 80% inhibition of the binding of ribosomes and their large (60S) subunit to the membrane. The inhibition was dependent on the amount of antibodies used, but comparable amounts of IgGs and Fab fragments from nonimmune serum had less effect. The p34 antibodies were also inhibitory for cotranslational translocation of secretory proteins, i.e. placental lactogen and serum albumin, across the membrane. These results suggest that p34 is involved in the binding of ribosomes to the microsomal membrane and that it is in close proximity to the protein translocation site in the microsomal membrane.

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Section: Inhibition Of Ribosome Binding By Antibodiesmentioning

confidence: 99%

“…The stripped microsomes possessed a number of binding sites, 65 nmol/g membrane proteins, as determined by the method of Yoshida et al [9]. The stripped microsomes (20/~g protein) were incubated with the indicated amounts of antibodies (see the legends to Figs.…”

Section: Inhibition Of Ribosome Binding By Antibodiesmentioning

confidence: 99%

Anti‐(p34 protein) antibodies inhibit ribosome binding to and protein translocation across the rough microsomal membrane

Ichimura¹,

Shindo²,

Uda³

et al. 1993

FEBS Letters

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“…Previous studies involving reconstitution of ribosome binding into liposomes have utilized lipids derived from a variety of sources and with varying compositions (5,10,11,21,22). In this study, we demonstrate that substantially different results are obtained depending on lipid compositions.…”

Section: Discussionmentioning

confidence: 69%

“…Our results presented here are consistent with these results, since we also found binding activity in pure PC liposomes that was not mediated by p180. A good candidate based on size and charge (8) would be p34, whose activity, coincidentally, was also characterized in pure PC liposomes (21). Based on the results of our p180 depletion studies, p34 probably has little if any activity in PS/PC or endogenous lipid liposomes.…”

Section: Discussionmentioning

confidence: 92%

Receptor-mediated Ribosome Binding to Liposomes Depends on Lipid Composition

Savitz¹,

Meyer²

1997

Journal of Biological Chemistry

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Ribosome binding to the endoplasmic reticulum has been traditionally studied using an in vitro assay in which potential ribosome receptors have been purified, incorporated into synthetic liposomes, and tested for activity. One such receptor (180 kDa; "p180") has been shown to bind ribosomes with high affinity in such a system when purified to homogeneity. This result has been challenged by data generated in other laboratories, and as a result, doubt has lingered as to the authenticity of p180 as a ribosome receptor. The contribution of the major difference between these studies, the lipid composition of the liposomes used in the in vitro assays, was assessed when identical fractions of rough endoplasmic reticulum-specific membrane proteins were incorporated into liposomes composed of only phosphatidylcholine (as used in other laboratories), a 50:50 mix of phosphatidylcholine and phosphatidylserine (as used in our original studies), or lipids derived from canine pancreatic microsomes (as a physiologically relevant control). The presence of PS was found to be crucial for the incorporation into and ribosome binding activity of p180 in liposomes. These observations are compatible with published studies on the importance of acidic phospholipids in ribosome binding to intact microsomes and reconcile the apparently conflicting in vitro results surrounding the assignment of p180 as a ribosome receptor.

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“…The difference in ribosome binding affinity between ER-derived reconstituted proteoliposomes and purified Sec61p-reconstituted membranes also leaves open the possibility that, in the former case, ribosome binding involves ER membrane proteins other than Sec61p. At least two ER membrane proteins with independent ribosome binding activity, p34 (42,43) and p180 (44), have been identified previously, although neither has been shown to associate with the translocon. Binding of ribosomes to membrane proteins other than Sec61p in reconstituted proteoliposomes might mask an effect of Sec61␤ depletion on the ribosome binding affinity of Sec61p itself.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Binding of Ribosomes to the β Subunit of the Sec61p Protein Translocation Complex

Levy

Kreibich

2001

Journal of Biological Chemistry

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The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the ␤ subunit of the complex (Sec61␤) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61␤ cytoplasmic domain (Sec61␤ c ) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61␤ c , like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61␤ c domain with ribosomal RNA. Like stripped ER membranes, the Sec61␤ c sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61␤ is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61␤, and it has been proposed that Sec61␤ assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61␤; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.The cotranslational translocation of proteins across the endoplasmic reticulum (ER) 1 membrane of eukaryotes involves the binding of translating ribosomes to receptor sites on the ER membrane, where the nascent protein enters the ER lumen through a proteinaceous pore. Translocation is preceded by a targeting phase in which ribosomes bearing pre-secretory nascent chains are selectively recruited to the ER membrane through the reciprocal action of at least two cytosolic factors,

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Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

Cited by 20 publications

References 29 publications

Anti‐(p34 protein) antibodies inhibit ribosome binding to and protein translocation across the rough microsomal membrane

Anti‐(p34 protein) antibodies inhibit ribosome binding to and protein translocation across the rough microsomal membrane

Receptor-mediated Ribosome Binding to Liposomes Depends on Lipid Composition

In Vitro Binding of Ribosomes to the β Subunit of the Sec61p Protein Translocation Complex

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