Abstract
The paramyxovirus HN polypeptide is a model type II membrane protein, containing an internal uncleaved signal/anchor (S/A) and is oriented in the membrane with an NH2-terminal cytoplasmic domain and COOH-terminal ectodomain (Ncyt topology). To test the role of NH2-terminal positively charged residues in directing the HN membrane topology, the 3 arginine (Arg) residues within the 17-amino-acid NH2-terminal domain were systematically converted to a glutamine or glutamate, and the topology of the mutant proteins was examined after expression in CV-1 cells. The data indicate that: (i) each of the NH2-terminal Arg residues contributes to the signal directing proper HN topology, since substitutions in any of the three positions resulted in approximately 13-23% inversion into the Nexo form; (ii) substitutions in the Arg directly flanking the signal/anchor domain resulted in slightly more inversion than those which were located more distally; and (iii) substitution with a negatively charged glutamate led to more inversion than did replacement with an uncharged glutamine. The effect of a single Arg to Glu substitution on the HN topology was enhanced when present in the context of a truncated NH2-terminal cytoplasmic tail (3 residues). A comparison of the sequences flanking the signal/anchor of well documented type III proteins showed that the majority of these proteins contain a negatively charged residue flanking the NH2-terminal side. An exception to this rule is the NB protein which contains a single positively charged Arg residue in this position. A chimeric protein containing the NB ectodomain and the HN S/A and HN ectodomain lead to a significant fraction (70%) of the chimeric protein adopting type II topology suggesting that the positive charge flanking the S/A domain is important for establishing type II topology. These data are discussed in the context of the loop model for the biogenesis of integral membrane proteins and the possible signals necessary for establishing differing orientations.
Highlights
From theHoward Huhes Medical Institute and Deportment of Biochemistry, Moleculcrr Biology and Cell Biology, Northwestern University, Euanston,IUinois 60208-3500
The data indicate that:(i)each of the NH4-terminalArg residues contributes to the signal directing pHroNpetor pology, since substitutionsin any of the threpeositionsresulted in -13-239'0 inversion into,. form; (ii)substitutions in the Arg directly flanking the signal/anchor domain resulted in slightly more inversion than those which were located more distally; and (iii)substitution characteristic membrane topology are contained within the amino acid sequence of the polypeptide (Blobel, 1980) and must be veryprecise as itappears that all naturally occurring membrane proteins adopt only a single final orientation
The majority of known membrane proteins which span the lipid bilayer a single time are classified as type I proteinsb, ased on the presence of both an NHz-terminal cleavable signal sequencewhich targets thenascent polypeptideto theER' membranethrough an interaction with the signal recognition particle (SRP; Walter and Lingappa, 1986) and a separate COOH-terminal hydrophobic domain which acts as a stop transfer domain
Summary
The N, or Nw topology have not been determined. Hydro- CeUs-Monolayer cultures of CV-1 cells were grown in Dulbecco’s phobicity appears to be the only structural requirement for modifiedEagle’s medium containing 10%fetal calf serum as described an uncleaved S/A to function in thetargeting and anchoring of a polypeptide (Audigieret al., 1987; Parks et al, 1989; Zerial et al, 1987). Isotopic Labeling of Polypeptides, Immunoprecipitation, N-Glycanme Digestions, Protease Treatment of Microsomal Membranes, and Polyacrylamide Gel Electrophoresis-Proteins were expressed in CV1cells as described (Parks andLamb, 1991) using a modified version of the vaccinia virus/?; RNA polymerase system of Fuerst et al. I1 proteins into the Nexoorientation, while the addition of positive charges to the COOH-terminal S/A-flanking region alone has little effect on topology (Beltzer et al, 1991; Parks and Lamb, 1991). TheHN Arg substitution mutants (Fig. 1) are denoted by a numbering system which is a continuation of that used from the S/A, as well as thecharge of the substitutingresidue, and the effect of these alterations is enhanced when in the context of a truncated NH2-terminal domain These results are discussed in a model for the topogenic signals of type I, 11, and 111proteins. N, and N, denote polypeptides with the WT HN anidnverted membrane orientations, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
