Identification of a ribosome receptor in the rough endoplasmic reticulum

Abstract

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.

Knockdown of p180 Eliminates the Terminal Differentiation of a Secretory Cell Line

“…Interestingly, p180 appears as a doublet in the blots, with the band of lower apparent molecular weight being prevalent during the period of p180 diminution. Previous in vitro studies have shown that p180 can exist as a functional 160-kDa proteolytic fragment (Savitz and Meyer 1990). It is possible that decreases in p180 mRNA and protein levels, as well as apparent molecular weight coincide with certain aspects of cell division, as our data on THP-1 cells show that most of the ER is initially restricted to the nuclear envelope, which disassembles during mitosis.…”

“…Originally isolated using a functional assay for ribosome binding, p180 has several distinctive features (Savitz and Meyer, 1990;Wanker et al, 1995). Anchored to the rough ER membrane by an N-terminal signal-anchor sequence of 28 amino acids, the remainder of p180 resides completely in the cytosol (Wanker et al, 1995).…”

“…Although the extent of membrane proliferation as well as the individual morphologies vary greatly, only one such system has yielded functional membranes whose proliferation coincided with increases in a cell's secretory capacity. This was achieved when a mammalian ribosome binding protein from the rough ER (p180) was expressed in yeast cells (Savitz and Meyer, 1990;Wanker et al, 1995;Becker et al, 1999). Proliferation of functional ER membranes was accompanied by a four-to fivefold increase in the secretion of a coexpressed protein into the medium (Becker et al, 1999).…”

“…Anchored to the rough ER membrane by an N-terminal signal-anchor sequence of 28 amino acids, the remainder of p180 resides completely in the cytosol (Wanker et al, 1995). It has high affinity (K d ϭ 10 -20 nm) for ribosomes (Savitz and Meyer, 1990), mediated by a domain in which a 10-amino acid consensus motif (NQGKKAEGAP) is repeated 54 times without interruption (Wanker et al, 1995). This domain is also needed for stabilization of mRNAs that encode proteins directed to the secretory pathway (Hyde et al, 2002 Abbreviations used: ER, endoplasmic reticulum; TPA, 12-O-tetradecanoylphorbol-13-acetate; THP-1, cell line derived from human acute monocytic leukemia; shRNA, small-hairpin RNA.…”

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

“…In one such study, Dejgaard and coworkers succeeded in separating and characterizing the native ribosome -translocon complex from canine pancreas microsomes (Dejgaard et al 2010). The native complex identified was larger than that predicted, and confirmed earlier work by Meyer and others (Hortsch et al 1986;Savitz and Meyer 1990), showing that the p180/ribosome receptor is indeed a constituent of the ribosometranslocon complex. Additional components such as CLIMP63, glucosidase I, and BAP31 were also assigned to the ribosome -translocon complex giving rise to a native size of 4 MDa rather than the previously predicted size of 2 MDa.…”