Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Transmembrane α-helices are the basic structural principle of most membrane proteins. As a result, the topology of multispanning membrane proteins is defined by a succession of helices of alternating orientation, separated by loops exposed to the cytoplasm and to the exoplasmic space [i.e., the lumen of the endoplasmic reticulum (ER) in eukaryotes or the exterior of the plasma membrane in prokaryotes (1)]. Upon integration, the transmembrane segments assemble to a helix bundle, while the loops fold in either the cytosol or the exoplasmic space, where chaperones may assist to prevent misfolding and aggregation.The integration of transmembrane domains and translocation of exoplasmic loops are mediated by the conserved Sec61/SecY translocon composed of Sec61-αβγ in the ER of eukaryotes and SecYEG in prokaryotes (2). The main subunit Sec61-α/SecY is a 10-transmembrane helix bundle that can open a pore across the membrane for the translocation of hydrophilic polypeptide chains and a lateral gate for the exit of hydrophobic segments into the lipid environment. In its idle state, the translocon is closed and stabilized by a short helix that forms an exoplasmic plug and by a central constriction.Proteins are targeted to the membrane by a hydrophobic signal sequence that, as it emerges from the ribosome, binds to signal recognition particle (SRP). The signal is either an amino-terminal cleavable signal peptide or the first transmembrane domain of the protein acting as an uncleaved signal anchor. SRP targets the translating ribosome to the SRP receptor and to the translocon. The ribosome binds to cytoplasmic loops of the C-terminal, half of the translocon, leaving a gap open toward the cytosol (3). As the protein is synthesized, its transmembrane segments are inserted successively into the lipid bilayer.Three distinct membrane integration processes can be distinguished (schematically shown in Fig. 1A). First, the signal (anchor) activates the translocon by intercalating between the gate helices and exiting toward the lipid phase (2). In the process, the hydrophilic flanking sequence is inserted into the pore for transfer into the lumen, and the plug is pushed away. This has been elucidated by a number of structures of translocons engaged with signal sequences (3–5). Which end of a signal anchor will be translocated is mainly determined by the flanking charges [the “positive–inside rule” states that the more positive end will be cytosolic (6–8)] and the folding state of the N-terminal domain hindering translocation (9). As the nascent chain is moving through the translocon pore, a hydrophobic transmembrane segment stops further transfer by leaving via the lateral gate into the lipid membrane as a stop-transfer sequence. The following polypeptide is then synthesized through the gap between ribosome and translocon into the cytosol until a further transmembrane segment again engages with the translocon, exits into the membrane, and inserts the downstream sequence into the pore. This reintegration process is similar to the initial signal integration. Furthermore, alternating stop-transfer and reintegration events may account for any number of transmembrane domains.Open in a separate windowFig. 1.Reintegration efficiency is affected by the cytoplasmic upstream sequence. (A) Schematic representation of the three distinct integration processes in the biogenesis of membrane proteins: signal (anchor) integration (red transmembrane segment) (1), stop-transfer integration (blue) (2), and reintegration (green) (3). Transmembrane domains do not necessarily fully enter the translocon, before exiting into the membrane, but may associate with lipids early on and glide along the gate, as proposed by Cymer et al. (43). (B) The construct RI-DP128 was derived from wt DPAPB via ST-DP128 (DPAPB-H in ref. 12), as shown schematically with the transmembrane domains in red, blue, and green as in A. Rings indicate potential N-glycosylation sites. A C-terminal triple–HA-tag is shown in gray. To analyze the hydrophobicity threshold of membrane integration, the H segment sequence GGPGAAAAAAAAAAAAAAAAAAAGPGG, with 0 to 19 alanines replaced by leucines, were inserted as a potential reintegration sequence (green) in RI-DP. Below, the DPAPB sequences of the cytosolic loop between the stop-transfer and the reintegration domains of RI-DP128 and the deletion constructs RI-DP100–22 are listed, with a dash indicating the deletion site. At least 20 residues preceding the reintegration H segment were kept constant in all constructs. (C) Schematic representation of the two topologies when the H segments do or do not initiate reintegration and C-terminal translocation. cyt, cytosol; exo, exoplasm = ER lumen. Glycosylations are indicated by Y. (D) The various constructs, each with different reintegration H segments composed of 19 alanines, with 0 to 19 of them replaced by leucine residues (L#), were expressed in yeast cells, labeled with [³⁵S]methionine for 5 min, immunoprecipitated, and analyzed by SDS–gel electrophoresis and autoradiography. Based on the glycosylation pattern, integration (I) or nonintegration (N) of the H segment, as well as uninserted unglycosylated products (U) were distinguished. To identify the position of the unglycosylated polypeptide, a sample was analyzed after deglycosylation by endoglycosidase H (H) on the right. (E) The fraction of products with reintegrated H segments as a percentage of the total membrane-integrated proteins was plotted versus the number of leucine residues in the H segment (mean, SD, and the individual values of at least three independent experiments). The curves are labeled with the length of the constructs’ cytosolic loops between stop transfer and H segment. (F) The cytoplasmic sequence between stop transfer and H segment is shown for RI-CP145 (residues 21 to 160 of pre-CPY, in gray) and RI-CP22 (residues 144 to 160). (G) The CPY constructs were expressed and analyzed, as in D. (H) Reintegration of the H segment was quantified, as in E (mean, SD, and the individual values of at least three independent experiments).The main characteristic of transmembrane segments is their overall hydrophobicity on a length of ∼20 residues that are required for an α-helix to span the apolar core of a bilayer. This was confirmed by systematic quantitative analysis of a large number of so-called H segments, mildly hydrophobic sequences of 19 residues, for their efficiency as stop-transfer sequences (10). From these experiments, a “biological hydrophobicity scale” for all 20 amino acids was derived. The dependence of integration efficiency on hydrophobicity is consistent with a purely thermodynamic equilibration process between the pore interior and the lipid environment for each peptide segment entering the translocon. This concept allowed for the estimation of apparent free energy contributions ∆G^aa_app of each amino acid to membrane integration. The contribution of particular amino acids also depended on their position in a manner reflecting the structure of the membrane (11): Polarizable residues, such as tryptophan or tyrosine, energetically favor the polarity interface between apolar fatty acyl and polar lipid head group regions, while polar and charged residues are energetically most unfavorable in the center of the bilayer, thus reducing integration efficiency.The equilibration model is further supported by mutagenesis of the translocon. The six residues forming the central constriction of the translocation pore are predominantly hydrophobic. Their mutation to more hydrophilic amino acids, to increase polarity and hydration inside the pore, reduced the hydrophobicity threshold for membrane integration considerably (12, 13), as expected for a partitioning process.From these findings emerges the concept that each sequence of ∼20 residues, based on its amino acids and their position within the segment, autonomously defines its propensity to integrate into the membrane. The data on the energetics of membrane integration was mostly collected for stop-transfer integration at the mammalian ER. Yet similar hydrophobicity dependence and hydrophobicity scales for integration were determined in other systems, such as for stop transfer in yeast (12, 14) or bacteria (15, 16), for reintegration in yeast and mammalian ER (17) and even for TIM23-mediated integration into the inner mitochondrial membrane (18). The biological hydrophobicity scale thus is considered to be universal and generally applicable to transmembrane segments, irrespective of their orientation, the organism, or the compartment. Accordingly, it is also used for topology prediction from primary sequences (19).However, the different systems that were analyzed also showed significant quantitative shifts in the hydrophobicity threshold for integration. A simple hydrophobicity series, as initially introduced by Hessa et al. (10), is produced with H segments consisting of 19 alanines, of which 0 to 19 are replaced by leucine. The threshold for 50% stop-transfer integration was determined to be 3.1 leucines using mammalian in vitro translation with dog pancreas microsomes and 3.8 leucines in baby hamster kidney cells (10). Further studies (summarized in ref. 1) measured thresholds between one and seven leucines [for example, 4.4 and 3.5 leucines in yeast (12, 14), 1.1 and 2.0 in Escherichia coli (15, 16), and 7.3 in HeLa cells (20)]. Using the ∆G_app values from Hessa et al. (11), this covers a range of ∼3 kcal/mol. Different lipid compositions and translocon sequences, but also the reporter constructs, may contribute. As to the latter, it has indeed been observed that the sequence immediately following an H segment can influence integration efficiency, most likely by conformationally hindering or facilitating stop-transfer release into the bilayer (21). This already challenged to some extent the autonomy by which each sequence determines its integration propensity.There is only one study that analyzed the hydrophobicity threshold of reintegration sequences (17). Surprisingly, it was found to be considerably lower than for stop-transfer integration with 0.9 versus 3.1 leucines in a mammalian in vitro translation/membrane insertion system and with 2.2 versus 4.4 leucines in yeast cells. In the present study, we set out to test the reintegration efficiency in Saccharomyces cerevisiae using the same H segments in a different reporter construct. Our results revealed a surprising dependence of reintegration on the length and/or characteristics of the sequence preceding the reintegration domain. A similar dependence was also found for stop-transfer integration. The data indicate that the folding properties of the cytoplasmic and luminal loop sequences in membrane proteins determine the integration efficiency of subsequent transmembrane domains.