Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.

Proline-induced disruption of a transmembrane alpha-helix in its natural environment.

alpha-Helix formation in globular proteins has been studied both theoretically and experimentally for decades, while a lack of both high-resolution structures and suitable experimental techniques has hampered the study of helices in membrane proteins. We have developed a new experimental approach, glycosylation mapping, where the active site of the lumenally exposed endoplasmic reticulum enzyme oligosaccharyl transferase is used as a point of reference against which the position of a transmembrane segment in the membrane can be measured. Here, we report an initial analysis of the helix-breaking properties of proline residues inserted in a transmembrane helix. We find that proline residues can break a transmembrane helix, but only when inserted near the end, and only when the helix is sufficiently long. The glycosylation mapping technique may be generally useful for determining the position of transmembrane helices in the membrane.

Elevated expression and genetic association links the SOCS3 gene to atopic dermatitis.

In a systematic analysis of global gene-expression patterns, we found that SOCS3 messenger RNA was significantly more highly expressed in skin from patients with atopic dermatitis than in skin from healthy controls, and immunohistochemical analysis confirmed a similar elevation of SOCS3 protein. Furthermore, we found a genetic association between atopic dermatitis and a haplotype in the SOCS3 gene in two independent groups of patients (P<.02 and P<.03). These results strongly suggest that SOCS3, located in a chromosomal region previously linked to the disease (17q25), is a susceptibility gene for atopic dermatitis.

Stop-transfer function of pseudo-random amino acid segments during translocation across prokaryotic and eukaryotic membranes.

We have measured the efficiency of stop-transfer function for a set of pseudo-random, 18-residue amino acid segments, both in Escherichia coli and in mammalian microsomes. In general, stop-transfer function correlates well with the mean hydrophobicity of the segment, though exceptions exist. Kinetic studies suggest that polar segments are rapidly translocated through the E. coli inner membrane and that strongly hydrophobic segments become permanently anchored, while sequences with an intermediate mean hydrophobicity become partly trapped in a transmembrane disposition for a considerable time before being released to the periplasm or degraded.

Divergent evolution of membrane protein topology: the Escherichia coli RnfA and RnfE homologues.

Although the molecular evolution of protein tertiary structure and enzymatic activity has been studied for decades, little attention has been paid to the evolution of membrane protein topology. Here, we show that two closely related polytopic inner membrane proteins from Escherichia coli have evolved opposite orientations in the membrane, which apparently has been achieved by the selective redistribution of positively charged amino acids between the polar segments flanking the transmembrane stretches. This example of divergent evolution of membrane protein topology suggests that a complete inversion of membrane topology is possible with relatively few mutational changes even for proteins with multiple transmembrane segments.

The internal repeats in the Na+/Ca2+ exchanger-related Escherichia coli protein YrbG have opposite membrane topologies.

We have determined the topology of the Escherichia coli inner membrane protein YrbG, a putative Na(+)/Ca(2+) exchanger with homology to a family of eukaryotic ion exchangers. Our results show that the two homologous halves of YrbG both have five transmembrane segments but opposite membrane orientations. This has implications for our understanding of the function of Na(+)/Ca(2+) exchangers and provides an example of "divergent" evolution of membrane protein topology.

SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context.

We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is inhibited. The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop in its normal context is only minimally affected by SecA inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins. Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition. Thus, SecA-dependence is not an all-or-none phenomenon and is not only a simple function of, e.g. the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments.

Membrane topology of the 60-kDa Oxa1p homologue from Escherichia coli.

We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule.

Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in Escherichia coli.

Assembly of several inner membrane proteins-leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep-has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC.

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec-translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo-crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.