Article

Topological Rules for Membrane Protein Assembly in Eukaryotic Cells

Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/1997; 272(10):6119-27. DOI: 10.1074/jbc.272.10.6119
Source: PubMed

ABSTRACT

Insertion into the endoplasmic reticulum membrane of model proteins with one, two, and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops has been studied both in vitro and in vivo. Membrane insertion of these same constructs has previously been analyzed in Escherichia coli, thus making possible a detailed comparison between the topological rules for membrane protein assembly in prokaryotic and eukaryotic cells. In general, we find that positively charged residues have similar effects on the membrane topology in both systems when they are placed in the N-terminal tail but that the effects of charged residues in internal loops clearly differ. Our results rule out a sequential start-stop transfer model where successive hydrophobic segments insert with alternating orientations starting from the most N-terminal one as the only mechanism for membrane protein insertion in eukaryotic cells.

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    • "Therefore, we concluded that the P4H enzyme responsible for post-translational rhEPO modification is located in the secretory compartments, i.e. the endoplasmic reticulum (ER) or the Golgi apparatus. All sequences display a hydrophobic segment near the N-terminus following a positively charged residue, suggesting a localization of the proteins in the secretory compartments40. However, we examined the subcellular localization of the seven moss P4Hs in silico with four different programs based on different algorithms. "
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    • "The insertion of proteins into membranes is thought to be achieved by a variety of conserved translocases and integrases (such as the well-described Sec translocon) acting both independently and cooperatively (Samuelson et al. 2000; Dalbey et al. 2011; Nishiyama et al. 2012). The addition of positive charges to the N-termini of transmembrane proteins can prevent the translocation of the termini across membranes in both E. coli and eukaryotes (Gafvelin et al. 1997), whether they require the main Sec protein-conducting channel (Li et al. 1988; Yamane and Mizushima 1988) or not (Whitley et al. 1994). Although the prevalence of the positive-inside rule is recognized , the mechanisms by which positive charges exert their topogenic effects are not well understood. "
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    • "To assess the effect of the presence of ionizable residues on the GpA TM segment insertion into biological membranes, we located this hydrophobic sequence (Fig. 4A) in place of the second TM fragment of the well-characterized Escherichia coli inner membrane protein leader peptidase (Lep). Although of bacterial origin, Lep integrates efficiently into dog pancreas microsomes with the same topology as in E. coli [30] (i.e., with both the N- and C-termini exposed to the luminal side of the ER membrane) and the presence of its first TM segment together with the cytoplasmic P1 domain (Figure 4B) is sufficient for proper targeting of chimeric proteins to the eukaryotic membrane [30], [31]. An engineered glycosylation site placed at the C-terminal P2 domain is glycosylated efficiently upon correct insertion into the microsomal membrane (Fig. 4B), serving as a reporter to distinguish between a lumenal (glycosylated) and a cytoplasmic (unglycosylated) location. "
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