FUNCTIONAL-CHARACTERIZATION OF THE 180-KD RIBOSOME RECEPTOR IN-VIVO

A cDNA encoding the 180-kD canine ribosome receptor (RRp) was cloned a nd sequenced. The deduced primary structure indicates three distinct d omains: an NH2-terminal stretch of 28 uncharged amino acids representi ng the membrane anchor, a basic region (pI = 10.74) comprising the rem ainder 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-a mino acid consensus motif, NQGKKAEGAP, repeated 54 times in tandem wit hout interruption in the NH2-terminal positively charged region. We po stulate that this repeated sequence represents a ribosome binding doma in which mediates the interaction between the ribosome and the ER memb rane. To substantiate this hypothesis, recombinant full-length ribosom e 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 biochem ical analyses showed all proteins were targeted to, and oriented corre ctly in the ER membrane. In vitro ribosome binding assays demonstrated that yeast microsomes containing the full-length canine receptor or o ne lacking the COOH-terminal domain were able to bind two to four time s as many human ribosomes as control membranes lacking a recombinant p rotein or microsomes containing a receptor lacking the NH2-terminal ba sic domain. Electron micrographs of these cells revealed that the expr ession of all receptor constructs led to a proliferation of perinuclea r ER membranes known as ''karmellae.'' Strikingly, in those strains wh ich expressed cDNAs encoding a receptor containing the putative riboso me binding domain, the induced ER membranes (examined in situ) were ri chly studded with ribosomes. In contrast, karmellae resulting from the expression of receptor cDNA lacking the putative ribosome binding dom ain were uniformly smooth and free of ribosomes. Cell fractionation an d biochemical. analyses corroborated the morphological characterizatio n. Taken together these data provide further evidence that RRp functio ns as a ribosome receptor in vitro, provide new evidence indicating it s functionality in vivo, and in both cases indicate that the NH2-termi nal basic domain is essential for ribosome binding.