IngMarie Nilsson

Fine-tuning the topology of a polytopic membrane protein: Role of positively and negatively charged amino acids

The effects of positively and negatively charged residues on the membrane topology of a model E. coli protein with two transmembrane segments have been studied. We show that addition or removal of as little as a single positively charged lysine residue in one of two critical regions can be sufficient to reverse the transmembrane topology of the molecule from Nout-Cout to Nin-Cin. Negatively charged residues are much less potent and significantly affect the topology only if present in high numbers. Finally, we provide data to suggest that sec-independent and sec-dependent translocation mechanisms differ in their sensitivity to positively charged amino acids.

Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits.

In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (α2, α5, β1, β2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe1129 in α2, to Phe1026 in α5, to Ile757 in β1, and to His728 in β2. The α-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys1125 in α2, Lys1022 in α5, Lys752 in β1, and Lys724 in β2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed.

A signal peptide with a proline next to the cleavage site inhibits leader peptidase when present in a sec‐independent protein

Proline residues are rarely found in the three most C‐terminal positions of bacterial signal peptides, and have never been found in position +1 immediately following the cleavage site. It was recently shown that a Pro+1 mutation in the E. coli maltose binding protein precursor not only prevents cleavage of the signal peptide but also inhibits the leader peptidase enzyme, resulting in cessation of cell growth (Barkocy‐Gallagher, G.A. and Bassford, P.J. (1992) J. Biol. Chem. (in press)). Since maltose binding protein is dependent on the sec machinery for translocation across the inner membrane, it was not clear if this ‘Pro+1’ effect was restricted to sec‐dependent proteins, or whether it applies also to proteins that do not require the sec functions for translocation. We now present data suggesting that the striking phenotypic effects of Pro+1 mutations can be elicited also by sec‐independent proteins.

Positively charged amino acids placed next to a signal sequence block protein translocation more efficiently in Escherichia coli than in mammalian microsomes

Positively charged amino acids are known efficiently to block protein secretion in Escherichia coli, when placed within a short distance downstream of a signal sequence. It is not known whether the same applies to protein secretion in eukaryotic cells, though statistical studies of signal sequences of prokaryotic and eukaryotic secretory proteins have suggested that the situation may be different in this case. Here, we show that identical charge mutations in a model protein have different effects on membrane translocation in E. coli and in mammalian microsomes, and that the ‘charge block’ effect is much more pronounced in the prokaryotic system. This finding has implications not only for our understanding of the mechanisms of protein secretion, but also points to a potential problem in the expression of eukaryotic secretory proteins in bacteria.

Insertion of a Bacterial Secondary Transport Protein in the Endoplasmic Reticulum Membrane

The sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) contains 12 hydrophobic potential transmembrane domains. Surprisingly, an alkaline phosphatase fusion study in Escherichia coli has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582–25589). To provide independent data on the topology and mode of membrane insertion of CitS, we have investigated its insertion into the endoplasmic reticulum (ER) membrane. By using in vitro translation of model proteins in the presence of dog pancreas microsomes, each of the putative transmembrane segments of CitS was assayed for its potency to insert into the ER membrane, both as an isolated segment as well as in the context of COOH-terminal truncation mutants. All 12 segments were able to insert into the membrane as Ncyt-Clumsignal anchor sequences. In a series of COOH-terminal truncation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segments VIII and IX, which, according to the alkaline phosphatase fusion study, are in the periplasm of E. coli, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segments and also demonstrate that the sequence requirements for signal anchor and stop transfer function are different.

Cleavage of a tail-anchored protein by signal peptidase

Tail‐anchored proteins are post‐translationally targeted and inserted into the endoplasmic reticulum membrane. They do not use the co‐translational signal‐recognition particle (SRP)‐dependent pathway, but rather utilize an ill‐defined, ATP‐dependent mechanism. Here, we show that a tail‐anchored protein can be cleaved by signal peptidase and that the sequence requirements for efficient cleavage seem to be the same as for cleavage of co‐translationally targeted SRP‐dependent proteins.

Calnexin can interact with N‐linked glycans located close to the endoplasmic reticulum membrane

Abstract Calnexin is a central component of the ‘quality control’ system in the endoplasmic reticulum (ER). Calnexin binds to monoglycosylated oligosaccharides on incompletely folded soluble and membrane proteins in the lumen of the ER and prevents exit from the organelle. We have previously found that the oligosaccharide transferase enzyme can add glycosyl moieties to a membrane protein when the acceptor site is as close as 12–13 residues away from the nearest transmembrane segment (J. Biol. Chem. 268, 5798). We now show that calnexin can bind to oligosaccharides located this close to the membrane, suggesting that its binding site is held at a similar distance from the membrane as is the active site of the oligosaccharide transferase. We further show that calnexin can bind efficiently to glycosylated but not to non‐glycosylated forms of a bacterial inner membrane protein, suggesting that it does not have a general affinity for non‐glycosylated proteins.

Different sec-requirements for signal peptide cleavage and protein translocation in a model E. coli protein

We describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non‐functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located ~30 residues downstream of the signal peptide. When this segment is deleted, normal SecA‐dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA‐independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.