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Functional characterization of the 180-kD ribosome receptor in vivo

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Item Type:Article
Title:Functional characterization of the 180-kD ribosome receptor in vivo
Creators Name:Wanker, E.E. and Sun, Y. and Savitz, A.J. and Meyer, D.I.
Abstract:A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned and sequenced. The deduced primary structure indicates three distinct domains: an NH2-terminal stretch of 28 uncharged amino acids representing the membrane anchor, a basic region (pI = 10.74) comprising the remainder of the NH2-terminal half and an acidic COOH-terminal half (pI = 4.99). The most striking feature of the amino acid sequence is a 10-amino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem without interruption in the NH2-terminal positively charged region. We postulate that this repeated sequence represents a ribosome binding domain which mediates the interaction between the ribosome and the ER membrane. To substantiate this hypothesis, recombinant full-length ribosome receptor and two truncated versions of this protein, one lacking the potential ribosome binding domain, and one lacking the COOH terminus, were expressed in Saccharomyces cerevisiae. Morphological and biochemical analyses showed all proteins were targeted to, and oriented correctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or one lacking the COOH-terminal domain were able to bind two to four times as many human ribosomes as control membranes lacking a recombinant protein or microsomes containing a receptor lacking the NH2-terminal basic domain. Electron micrographs of these cells revealed that the expression of all receptor constructs led to a proliferation of perinuclear ER membranes known as "karmellae." Strikingly, in those strains which expressed cDNAs encoding a receptor containing the putative ribosome binding domain, the induced ER membranes (examined in situ) were richly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding domain were uniformly smooth and free of ribosomes. Cell fractionation and biochemical analyses corroborated the morphological characterization. Taken together these data provide further evidence that RRp functions as a ribosome receptor in vitro, provide new evidence indicating its functionality in vivo, and in both cases indicate that the NH2-terminal basic domain is essential for ribosome binding.
Keywords:Amino Acid Sequence, Base Sequence, Molecular Cloning, DNA Primers, Endoplasmic Reticulum, Gene Expression, Hela Cells, Intracellular Membranes, Molecular Sequence Data, Messenger RNA, Cytoplasmic and Nuclear Receptors, Nucleic Acid Repetitive Sequences, Ribosomes, Saccharomyces cerevisiae, Animals, Dogs
Source:Journal of Cell Biology
ISSN:0021-9525
Publisher:Rockefeller University Press
Volume:130
Number:1
Page Range:29-39
Date:July 1995
Additional Information:Copyright (c) 1995 by The Rockefeller University Press
Official Publication:http://www.jcb.org/cgi/content/abstract/130/1/29
PubMed:View item in PubMed

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