Functional characterization of the 180-kD ribosome receptor in vivo.

Abstract

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, NQGK-KAEGAP, 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.

IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast

“…The expression of different regions of canine p180 in yeast gives rise to rough or smooth ER-like membranes as previously documented primarily by electron microscopy as well as biochemically through measurement of increase in membrane lipid on a per cell basis (Wanker et al, 1995). To demonstrate that the membranes observed resulted from the synthesis of new membranes and not from the incorporation of proteins into preexisting membranes, total membrane quantification was carried out.…”

“…Membrane proliferation has been observed in cells that express high levels of certain integral ER membrane proteins such as the yeast HMG-CoA reductase isozymes, Hmg1p and Hmg2p (Wright et al, 1988;Koning et al, 1996), cytochrome P450 (Schunck et al, 1991), and various domains of the mammalian ribosome receptor, p180 (Wanker et al, 1995;Becker et al, 1999). ER-like membrane morphologies arise from the overexpression of the peroxisomal integral membrane protein, Pex15p (Elgersma et al, 1997).…”

“…To identify genes whose levels of expression change during stimulated membrane production, microarray analysis was performed using mRNA isolated from cells expressing the canine ribosome receptor (p180) or specific regions of it known to produce membrane proliferation in yeast. The canine ribosome receptor is an integral membrane protein of the endoplasmic reticulum (ER) consisting of three distinct regions based on its amino acid sequence (Wanker et al, 1995). Briefly, full-length p180 (FL) consists of an amino terminal membrane-anchoring domain, a basic region consisting of 54 tandem decapeptide repeats involved in ribosome binding and a C-terminal predicted coiled-coil domain of unknown function (Langley et al, 1998).…”

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

“…Specific endoplasmic reticulum (ER) membrane arrays are induced in yeast by increasing the levels of certain ER membrane proteins, such as 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase (Wright et al, 1988), cytochrome P450 (Schunck et al, 1991), cytochrome b 5 (Vergeres et al, 1993), Sec12p (Nishikawa et al, 1994), the canine ribosome receptor (Wanker et al, 1995), and Pbn1p (protease B negative 1 protein) (Naik and Jones, 1998). Analysis of these inducible membranes provides an opportunity to discover the molecular mechanisms cells use to regulate the synthesis and organization of new membrane arrays.…”

Proliferation of the endoplasmic reticulum occurs normally in cells that lack a functional unfolded protein response

“…A dramatic example of regulation of organelle biogenesis involves the proliferation of ER arrays in response to increased expression of certain ER membrane proteins (Naik and Jones, 1998;Nishikawa et al, 1994;Schunck et al, 1991;Vergeres et al, 1993;Wanker et al, 1995;Wright et al, 1988). When the level of the sterolbiosynthetic enzyme, HMG-CoA reductase, is elevated experimentally or by treatment with drugs (Singer et al, 1988), cells assemble novel arrays of smooth ER.…”