N-Tail translocation in a eukaryotic polytopic membrane protein Synergy between neighboring transmembrane segments Magnus Monne  , Guro Gafvelin*, Robert Nilsson and Gunnar von Heijne Department of Biochemistry, Stockholm University, Sweden We have used the natural N-glycosylation site in the N-tail of cig30, a eukaryotic polytopic membrane protein, as a marker for N-tail translocation across the microsomal membrane. Analysis of C-terminally truncated cig30 constructs reveals that the first transmembrane segment is sufficient for translocation of the wild-type N-tail; in contrast, in a mutant with four arginines introduced into the N-tail the second transmembrane segment is also required for efficient N-tail translocation. Our observations imply a non-sequential assembly mechanism in which the ultimate location of the N-tail relative to the membrane may depend on more than one transmembrane segment. Keywords: cig30; membrane protein assembly; N-tail translocation; topology. Translocation of proteins across the membrane of the endo- plasmic reticulum (ER) is normally initiated by an N-terminal signal peptide or signal-anchor sequence [1], resulting in translocation of the downstream part of the nascent chain. Many integral membrane proteins do not follow this simple N- to C-terminal translocation mechanism, however, but rather have their polar N-terminal tails (N-tails) translocated across the membrane into the lumen of the ER; this is the case for, e.g. most of the G-protein-coupled receptors [2]. For such proteins, the most N-terminal transmembrane (TM) segment is thought to function as a `reverse signal-anchor sequence' [1] that targets the protein to the ER translocon via the normal signal recog- nition particle (SRP) pathway, but then inserts across the membrane with its N-terminal rather than C-terminal flanking domain facing the lumen. The net charge difference across the N-terminal TM [3], the length of this TM [4], and the folding properties of the N-tail domain [5] are all known to affect the efficiency of N-tail translocation. According to the simplest model of how multispanning (polytopic) membrane proteins insert cotranslationally into the ER membrane [6], the hydrophobic TM segments serve alter- nately as start and stop transfer signals, and membrane insertion is thus viewed as a sequential process starting from the N-terminal TM. While this model, based on the notion of independently acting topogenic signals, may account for the assembly of some polytopic proteins, there are a number of counter examples where a given TM segment will only insert properly in the presence of its neighboring TMs but not otherwise [7±12], implying that topogenic information present in more than one TM must be `decoded' simultaneously. In this paper, we address the question of whether the most N-terminal TM in a polytopic eukaryotic protein, cig30 (cold-inducible glycoprotein of 30 kDa) [13], is by itself sufficient to induce translocation of the <35 residues long, lumenally oriented N-tail, or if additional topogenic informa- tion is needed. We find that the first TM is indeed the only hydrophobic segment that is required for efficient translocation of the N-tail, but that membrane targeting is inefficient unless TM1 is followed by a sufficiently long stretch of chain. In contrast, in a cig30 mutant in which additional positively charged residues have been inserted into the N-tail, efficient translocation is only observed when two transmembrane segments are present, and only if they are followed by a sufficiently long polar domain. These results indicate that topogenic information present in the entire N-tail±TM1±TM2 region can affect the topology of the protein, implying a non- sequential assembly mechanism in which the ultimate location of the N-tail may depend on more than one TM segment. MATERIALS AND METHODS Enzymes and chemicals Unless otherwise stated, all enzymes were from SDS Promega (Falkenberg, Sweden). Ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides, the cap analog m7G(5 0 )-ppp(5 0 )G, T7 DNA polymerase, and [ 35 S]methionine were from Amersham- Pharmacia Biotech (Uppsala, Sweden). Plasmid pGEM1, dithiothreitol, BSA, RNasine, rabbit reticulocyte lysate, and the RiboMAX TM system were from SDS Promega. Spermidine and phenylmethanesulfonyl fluoride were from Sigma. Oligo- nucleotides were from Cybergene (Stockholm, Sweden). Proteinase K was from Life Technologies, Inc. DNA techniques SalI and XbaI restriction sites were introduced by PCR at the 5 0 and 3 0 ends of the cig30 gene, respectively. The PCR fragment was cloned into phage M13 mp18 and into a pGEM1-derived plasmid after a modified upstream region of the lepB gene [14] containing a `Kozak consensus sequence' for efficient ribosome binding [15]. Site-directed mutagenesis was performed as described by Kunkel and Geissloder [16,17] to introduce 4 arginine codons after the 9th or 28th codon in the cig30 coding Eur. J. Biochem. 263, 264±269 (1999) q FEBS 1999 Correspondence to G. von Heijne, Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden. Fax: +46 8 15 36 79, Tel.: + 46 8 16 25 90, E-mail: gunnar@biokemi.su.se Abbreviations: ER, endoplasmic reticulum; TM, transmembrane; SRP, signal recognition particle; cig30, cold-inducible 30-kDa glycoprotein. *Present address: Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska Hospital, S-171 76 Stockholm, Sweden. (Received 8 February 1999; revised 29 March 1999: accepted 22 April 1999)