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. 2017 Apr 1;130(7):1307-1320.
doi: 10.1242/jcs.198655. Epub 2017 Feb 20.

Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

Michael McKenna  1 , Rachel E Simmonds  2 , Stephen High  3
Affiliations

Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

Michael McKenna et al. J Cell Sci. .

Abstract

Mycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression that are characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, the translocation of TMP lumenal domains driven by an N-terminal cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation that is driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs.

Keywords: Endoplasmic reticulum; Membrane protein; Mycobacterium ulcerans; Mycolactone; Protein translocation; Sec61.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
TMDs of the type I TMPs CD3δ and GypA can partially rescue their membrane integration in the presence of mycolactone. (A) CD3δ constructs (wild type and D111L mutant) used in this study. (B) Phosphorimage of CD3δ that had been in vitro translated in the absence or presence of mycolactone (MYC) and then treated with or without Endoglycosidase H (EndoH). Glycosylated (‘+g’) and non-glycosylated (‘0g’) substrate is indicated. (C) Graph showing the reduction in the amount of ‘+g’ CD3δ and related constructs in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. Statistical test performed was one-way ANOVA. Error bars show mean±s.d. CD3δ, n=9; CD3δΔTMD, n=3; CD3δD111L, n=7. ns, P>0.05; *P≤0.05; **P≤0.01, ***P≤0.001. (D) Translation of the secretory protein pre-prolactin (PPL) in the absence and presence of mycolactone shown for comparative purposes. Non-cleaved (‘nc’) and signal cleaved (‘sc’) substrate is indicated. (E) Translation of CD3δΔTMD in the absence or presence of mycolactone. (F) Estimated TMD hydrophobicity values (kcal/mol) of CD3δ and CD3δD111L. Hydrophobicity is based on free energy (ΔG) values, calculated using http://dgpred.cbr.su.se/ (Hessa et al., 2007). (G) Translation of CD3δD111L in the absence or presence of mycolactone.
Fig. 2.
Fig. 2.
ER integration of the type I TMP CD3δ in the presence of mycolactone is driven by its TMD. (A) Truncated mRNAs coding for CD3δ (top panel) and CD3δD111L (bottom panel) and lacking stop codons translated in the absence or presence of mycolactone (MYC) without puromycin-mediated release. The nascent chain length of each truncation is shown, as well as the number of residues synthesised C-terminal to the TMD to provide an estimate of its distance from the peptidyl-transferase centre (PTC) of the ribosome. Truncations where all or part of the TMD is likely obscured by the ribosomal exit tunnel (based on Cabrita et al., 2016) are indicated by the bracketed area. CD3δ158 is encompassed by a dashed bracket, since its TMD is likely on the border of having just fully emerged from the ribosomal exit tunnel. Arrowheads indicate maximal glycosylation resulting from the TMD-dependent rescue of integration in the presence of mycolactone. (B) Versions of (i) CD3δ and (ii) CD3δD111L lacking signal sequences (ΔSS) translated in the absence and presence of mycolactone, without or without subsequent EndoglycosidaseH (EndoH) treatment. (C) Predicted mechanism of type I TMP integration in the absence (i) and presence (ii) of mycolactone. Other symbols are as defined in Fig. 1 legend. ‘+g’, glycosylated; ‘0g’, non-glycosylated; ‘C’, C-terminus; FL, full length; ‘N’, N-terminus.
Fig. 3.
Fig. 3.
The large N-terminal domain of the type I TMP VCAM1 results in a complete block of its membrane integration by mycolactone. (A) VCAM1 translated in the absence or presence of mycolactone (MYC) and treated with EndoglycosidaseH (EndoH). (B) VCAM1 and VCAM160 constructs (wild type and S707L/S707L* mutants) used in this study. (C) VCAM160 and a version containing an artificial N-glycosylation site (C52N) translated in the absence or presence of mycolactone, without or without EndoH. (D) VCAM160 and a variant with a more hydrophobic TMD (VCAM160 S707L*) translated in the absence or presence of mycolactone, without or without subsequent EndoH treatment. Estimated TMD hydrophobicities (kcal/mol) are indicated in D. Graph shows the reduction in the amount of ‘+g’ VCAM160 and VCAM160 S707L* in the presence of mycolactone, relative to control samples, as described in the legend to Fig. 1. is also shown in D (graph). The statistical test performed was one-way ANOVA. Error bars show mean±s.d. VCAM1, n=3 VCAM160, n=4; VCAM160 S707L*, n=3). P-values are as defined in Fig. 1 legend. (E) Translation of VCAM1, VCAM160 and the secretory protein cecropin, possessing a C-terminal opsin tag (CecOPG2), performed with increasing concentrations of CAM741 or an equivalent volume of DMSO (‘−’). (F) VCAM160 and VCAM160 S707L* translated in the absence or presence of 250 nM CAM741. Other symbols are as defined in Fig. 1 legend.
Fig. 4.
Fig. 4.
Mycolactone does not interfere with type III TMP integration. (A) Translation of GypC in the absence or presence of mycolactone (MYC), followed by subsequent treatment with EndoglycosidaseH (EndoH). (B) Graph shows change in the amount of glycosylated (+g) GypC and related constructs in the presence of mycolactone, relative to control samples as described in the legend to Fig. 1. The statistical test performed was one-way ANOVA. Error bars show mean±s.d. GypC, n=10; others, n=3. Ns, not significant. (C) Estimated TMD hydrophobicities (kcal/mol) of GypC and related constructs. (D) Translation of two variants of GypC with reduced TMD hydrophobicity. (E) GypC truncations lacking stop codons. For crosslinking experiments, truncations contained a single artificially introduced cysteine residue at either position 52 or 84, as denoted by an asterisk. (F) Truncated GypC chains synthesised in the absence or presence of mycolactone without puromycin-mediated release. The glycosylation of nascent chains when still attached to the ribosome (indicated by ‘peptRNA’) was observed. (G) Truncated GypC chains containing a single cysteine residue [either *(52) or *(84)] synthesised in the absence or presence of mycolactone without puromycin-mediated release to generate membrane integration intermediates. Samples were treated with the crosslinking reagent BMH, subjected to extraction with alkaline sodium carbonate, and analysed by SDS-PAGE. Adducts between the nascent chain and Sec61β (xSec61β) or the nascent chain and Sec61α/Sec61α and Sec61β (xSec61α/αβ) are indicated (see also Fig. S3B). Mycolactone-sensitive adducts are indicated by arrowheads. Other symbols are as defined in Fig. 1 legend. FL, full length.
Fig. 5.
Fig. 5.
Mycolactone efficiently blocks type II TMP integration. (A) Full-length Ii (wild type and G47L Q48L mutant) and the Ii125 truncation used in this study. (B) Estimated TMD hydrophobicities (kcal/mol) of Ii and IiG47L Q48L. (C) Graph shows the reduction in the amount of glycosylated (+g) Ii and related constructs in the presence of mycolactone (MYC), relative to control samples as described in the legend to Fig. 1. The statistical test performed was one-way ANOVA. Error bars show mean±s.d. (n=3). P-values are as defined in Fig. 1 legend. Translation in the absence or presence of mycolactone performed using Ii (D), IiG47L Q48L (E) and Ii125 (F), which was followed by treatment with EndoglycosidaseH (EndoH). (G) Ii truncations used in this study. For crosslinking experiments, truncations contained either a native cysteine residue (C28) or one that was artificially introduced [*(50)]. A truncated version of TNFα used for crosslinking analysis (as described in MacKinnon et al., 2014) is shown for comparative purposes. Crosslinking was performed on Ii truncations (H) and Ii125*(50) (I) and the resulting adducts are labelled as described in the Fig. 4G legend. Other symbols are as defined in Fig. 1 legend. Puro, puromycin.
Fig. 6.
Fig. 6.
Mycolactone sensitivity is dependent upon which TMD-flanking region is translocated. (A) A chimeric protein containing Ii downstream of a pre-prolactin (PPL) signal sequence (i) and the two topologies it might assume following integration into RMs, depending on whether the region that is translocated is N-terminal (ii) or C-terminal (iii) of the TMD. (B) Translation of PPL-Ii and PPL-IiG47L Q48L* in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Samples were analysed following immunoprecipitation of Ii. (C) Graph showing the amount of signal-cleaved (‘sc’) or glycosylated (‘+g’) substrate in the presence of mycolactone relative to control samples. These values were determined by dividing the quantity of ‘sc’ or ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘sc’ or ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. (n=3). P-values and other symbols are as defined in Fig. 1 legend.
Fig. 7.
Fig. 7.
Mycolactone traps headfirst-inserting type II TMPs in an N-lumenal–C-cytosolic topology. (A) ASGPR H1 and ASGPR H1Δ. Translation of ASGPR H1 (B) and ASGPR H1Δ (C) performed in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Membrane fractions were subjected to extraction with alkaline sodium carbonate prior to analysis. (D) Graph shows the amount of glycosylated (‘+g’) and non-glycosylated (‘0g’) ASGPR H1 and ASGPR H1Δ in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ or ‘0g’ substrate obtained in the presence of mycolactone by the quantity of '+g' or '0g' substrate obtained in the absence of mycolactone and are expressed as percentages. Dashed red line represents the value for comparative material for samples treated with a vehicle control. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. (n=3). P-values are as defined in Fig. 1 legend. (E) Diagram showing type II TMPs that insert using a hairpin mechanism (i) or a headfirst/inversion mechanism (ii), as well as the headfirst insertion of type III TMPs (iii). Faded steps represent those that are prevented by mycolactone. Dashed arrow shows the predicted route taken by headfirst-inserting type II TMPs when inversion is prevented by mycolactone.
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