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First published online 24 April 2007
doi: 10.1242/jcs.002410

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Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones

Benjamin M. Abell^1,*, Catherine Rabu¹, Pawel Leznicki¹, Jason C. Young² and Stephen High^1,

¹ Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
² Department of Biochemistry, McGill University, Room 914, McIntyre Building, 3655 Promenade Sir William Osler, Montreal, QC, H3G 1Y6, Canada

View larger version (15K): [in this window] [in a new window]   Fig. 1. Model TA proteins. Sequences of the tail-anchor regions and C-terminal extensions of the polypeptides used in this study. Potential transmembrane (TM) domains are underlined, dots indicate hydrophilic domains extending beyond the sequence presented. Numbers in superscript show the total length of the polypeptides; –TM indicates replacement of the hydrophobic TM domain; G indicates a chimera with a C-terminal N-glycosylation site; OPG indicates a chimera with a C-terminal extension derived from bovine opsin including an N-glycosylation site; N indicates N-glycosylation-target residues.

View larger version (35K): [in this window] [in a new window]   Fig. 2. N-glycosylation indicates ATP-dependent post-translational integration. (A) mRNA encoding the full-length Sec61βG polypeptide but lacking a stop codon to terminate protein synthesis (see Fig. 1) was translated for 20 minutes, and nascent-chain release synchronised by the addition of puromycin. Incubation was continued in the presence of microsomes for 30 minutes and one sample was treated with EndoH. Glycosylated and non-glycosylated Sec61β are indicated (1g and 0g respectively). Quantification showed that, in the absence of EndoH treatment, 16% of the membrane-associated chains remaining after extraction with alkaline sodium carbonate solution were N-glycosylated. Molecular mass is indicated on the left (in kDa). (B) Sec61βG was released from isolated RNCs by puromycin treatment in the presence or absence of reticulocyte lysate (RL), then treated with or without 10 mM EDTA, followed by treatment with or without 10 mM Mg(OAc)2 as shown. Samples were finally incubated with microsomes for 30 minutes and membrane-associated material was isolated by extraction with alkaline sodium carbonate solution. Of the membrane-associated products recovered, 8% were N-glycosylated for the control sample (lane 4). Lower molecular weight forms of non-glycosylated Sec61β were more prevalent after RNC preparation (lanes 1-6, product 0g and below), most likely as a result of ribosome stacking (Ismail et al., 2006). We confirmed that EDTA treatment does not prevent N-glycosylation per se (data not shown), hence, a lack of glycosylated Sec61β reflects a lack of integration. (C) Sec61βG was released from isolated RNCs by puromycin treatment in the presence of buffer, reticulocyte lysate (RL) or lysate depleted of small molecules by gel filtration (Dep. RL), with additional ATP (A) or GTP (G) as shown. In one case, a double quantity of normal lysate was added (++). Samples were incubated with microsomes for 30 minutes and the membrane fraction was recovered after extraction with alkaline sodium carbonate solution as for B. The resulting material corresponding to non-glycosylated polypeptides (0g) and glycosylated polypeptides (1g) was quantified and standardised to the sample incubated with reticulocyte lysate (lane 1, relative integration=100). In this case, 6% of the membrane-associated products recovered were N-glycosylated for the control sample (lane 1).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Sec61β associates with cytosolic chaperones. (A) Sec61β was synthesised as in described for Fig. 2 and polypeptide chains were released from the ribosome by puromycin treatment. Samples were treated with apyrase to deplete nucleotide triphosphates, crosslinking reagents were added as shown and the resulting products resolved by SDS-PAGE. The location of Sec61β chains and the approximate molecular mass of major adducts are indicated (in kDa). (B) Products of SMCC cross-linking were subjected to immunoprecipitation with antisera recognising specific cytosolic components or a non-related serum (NRS). Adducts with SRP54 (filled circle) and Hsp40 (star) are shown. (C) Sec61β (lanes 1 to 5) or a version without the hydrophobic TM region, Sec61β-TM (lanes 6 to 10), were synthesised as for A) and total products analysed either before (lanes 1 and 6) or after (lanes 2 and 7) SMCC mediated cross-linking. Adducts were identified by immunoprecipitation carried out in the absence of prior SDS denaturation and using antisera specific for either Sec61β (lanes 4 and 9) or Hsc70 (lanes 5 and 10). A non-related serum was used as a control (lanes 3 and 8), adducts with Hsc70 are identified (filled square).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Specific chaperones stimulate the membrane integration of TA proteins. (A) Sec61βG was released from isolated RNCs by puromycin treatment for 5 minutes in the presence of ATP (except for –ATP) and various molecular chaperones or reticulocyte lysate (RL) as shown. Samples were incubated with ER-derived microsomes (K-RM) for 30 minutes, and membrane-associated material was isolated as before. N-glycosylated material was quantified after extraction with alkaline sodium carbonate solution and standardised relative to the sample incubated with reticulocyte lysate (set to 100). Of the membrane associated products recovered, 10% were N-glycosylated for the control sample (lane 12). (B) Sec61βOPG was used to analyse the role of molecular chaperones as described in A. In this case, the membrane-associated material was analysed directly after the isolation of the membrane fraction through a high-salt sucrose cushion because a comparison with subsequent alkaline extraction revealed that the two procedures give similar results with this precursor (supplementary material Fig. S3B). Combinations of chaperones were added together with ATP and integration efficiency was analysed on the basis of N-glycosylation efficiency in four independent experiments. One such experiment is presented together with the average level of stimulation for the different treatments and the ±s.e.m. For the experiment shown, 42% of the membrane-associated products recovered were N-glycosylated when the sample was incubated with reticulocyte lysate (lane 6). **P<0.01 for these chaperone combinations causing a stimulation of membrane integration when compared to the control (lane 7). (C) Sec61βG was treated as described for A, except that varying concentrations of CBAG, a C-terminal fragment of Bag1, were included. N-glycosylation was used to measure membrane integration, and the values were standardised relative to those obtained with Hsp40 and Hsc70 alone.

View larger version (25K): [in this window] [in a new window]   Fig. 5. Chaperone-mediated pathways operate in parallel with SRP-mediated targeting. (A) Sec61βG was released from isolated RNCs by puromycin treatment in the presence of reticulocyte lysate (), purified Hsp40 and Hsc70 with ATP () or purified SRP with GTP (). Samples were incubated with membranes for 0 to 30 minutes, membrane-associated material resistant to extraction with alkaline sodium carbonate solution was recovered as described above, and relative integration efficiency measured by N-glycosylation was compared with the value obtained with reticulocyte lysate after 30 minutes. (B) Sec61βG or Syb2G were isolated as RNCs and the polypeptides released from the ribosomes by puromycin treatment in the presence of reticulocyte lysate, or depleted reticulocyte lysate supplemented with SRP, ATP and GTP, as indicated. Samples were then incubated with membranes for up to 30 minutes and relative integration was measured by N-glycosylation as compared to the level obtained with reticulocyte lysate after 30 minutes.

View larger version (30K): [in this window] [in a new window]   Fig. 6. A combination of SRP and molecular chaperones mediate TA protein integration. The binding of SRP to TA proteins occurs at an early stage of biosynthesis, shortly after the nascent chain is released from the ribosome. If the nascent TA protein is a poor substrate for SRP, it can use the chaperone-mediated pathway. The Hsc70-Hsp40 combination mediates the major ATP-dependent route and their activity can be modulated by co-chaperones such as BAG1. The identity of the membrane component(s) to which the molecular chaperones deliver their substrates remains unclear (High and Abell, 2004).

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