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Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

Campbell JH, Hoang T, Jelokhani-Niaraki M, Smith MD - BMC Biochem. (2014)

Bottom Line: Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes.Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, ON, N2L 3C5, Canada. camp0410@mylaurier.ca.

ABSTRACT

Background: The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane.

Results: We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.

Conclusion: Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

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Related in: MedlinePlus

Design of atTic20, its truncated mutant and extramembrane N-terminal peptide. (A) Schematic representation of different forms of atTic20 used in this study. A cDNA encoding pre-atTic20 (including the transit peptide) was used to generate constructs encoding mature atTic20 and atTic20ΔN20, a truncated mutant lacking the 20 amino acid N-terminal domain (NTD), with C-terminal hexahistidine tags (6His). A 21-amino acid peptide corresponding to the NTD was also synthesized (amino acid sequence shown). (B) Results of in silico analyses suggest that atTic20 contains four transmembrane domains and that its N-terminal peptide is disordered. (i) IUPred was used for disorder predictions of atTic20. Transmembrane prediction for atTic20 was based on (ii) hydrophobicity plot and (iii) TMHMM analysis. Refer to the Methods for further detailed method and analysis.
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Fig1: Design of atTic20, its truncated mutant and extramembrane N-terminal peptide. (A) Schematic representation of different forms of atTic20 used in this study. A cDNA encoding pre-atTic20 (including the transit peptide) was used to generate constructs encoding mature atTic20 and atTic20ΔN20, a truncated mutant lacking the 20 amino acid N-terminal domain (NTD), with C-terminal hexahistidine tags (6His). A 21-amino acid peptide corresponding to the NTD was also synthesized (amino acid sequence shown). (B) Results of in silico analyses suggest that atTic20 contains four transmembrane domains and that its N-terminal peptide is disordered. (i) IUPred was used for disorder predictions of atTic20. Transmembrane prediction for atTic20 was based on (ii) hydrophobicity plot and (iii) TMHMM analysis. Refer to the Methods for further detailed method and analysis.

Mentions: Tic20 has been predicted to have 4 TM helices [1,10,12,15], and our in silico modelling using TopPred, 3D Distill servers and TMHMM are consistent with this prediction (Figure 1, Bii and Biii) [25,26]. The predictions are also consistent with atTic20 having small N- and C-terminal soluble segments [13], which we then examined for predicted intrinsic disorder using IUPred [27]. Using the short disorder parameter, IUPred predicted that the N-terminal segment has a strong tendency towards disorder (Figure 1Bi). Based on this analysis, we devised two constructs for further work: one encoding a mature, transit peptide-lacking version of atTic20 and an N-terminally truncated version (atTic20ΔN20) lacking the N-terminal amino acids of the mature protein that were predicted to form the intrinsically disordered segment (Figure 1A). Additionally, we prepared a synthetic peptide corresponding to the extramembrane N-terminal domain that was predicted to be disordered.Figure 1


Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

Campbell JH, Hoang T, Jelokhani-Niaraki M, Smith MD - BMC Biochem. (2014)

Design of atTic20, its truncated mutant and extramembrane N-terminal peptide. (A) Schematic representation of different forms of atTic20 used in this study. A cDNA encoding pre-atTic20 (including the transit peptide) was used to generate constructs encoding mature atTic20 and atTic20ΔN20, a truncated mutant lacking the 20 amino acid N-terminal domain (NTD), with C-terminal hexahistidine tags (6His). A 21-amino acid peptide corresponding to the NTD was also synthesized (amino acid sequence shown). (B) Results of in silico analyses suggest that atTic20 contains four transmembrane domains and that its N-terminal peptide is disordered. (i) IUPred was used for disorder predictions of atTic20. Transmembrane prediction for atTic20 was based on (ii) hydrophobicity plot and (iii) TMHMM analysis. Refer to the Methods for further detailed method and analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307631&req=5

Fig1: Design of atTic20, its truncated mutant and extramembrane N-terminal peptide. (A) Schematic representation of different forms of atTic20 used in this study. A cDNA encoding pre-atTic20 (including the transit peptide) was used to generate constructs encoding mature atTic20 and atTic20ΔN20, a truncated mutant lacking the 20 amino acid N-terminal domain (NTD), with C-terminal hexahistidine tags (6His). A 21-amino acid peptide corresponding to the NTD was also synthesized (amino acid sequence shown). (B) Results of in silico analyses suggest that atTic20 contains four transmembrane domains and that its N-terminal peptide is disordered. (i) IUPred was used for disorder predictions of atTic20. Transmembrane prediction for atTic20 was based on (ii) hydrophobicity plot and (iii) TMHMM analysis. Refer to the Methods for further detailed method and analysis.
Mentions: Tic20 has been predicted to have 4 TM helices [1,10,12,15], and our in silico modelling using TopPred, 3D Distill servers and TMHMM are consistent with this prediction (Figure 1, Bii and Biii) [25,26]. The predictions are also consistent with atTic20 having small N- and C-terminal soluble segments [13], which we then examined for predicted intrinsic disorder using IUPred [27]. Using the short disorder parameter, IUPred predicted that the N-terminal segment has a strong tendency towards disorder (Figure 1Bi). Based on this analysis, we devised two constructs for further work: one encoding a mature, transit peptide-lacking version of atTic20 and an N-terminally truncated version (atTic20ΔN20) lacking the N-terminal amino acids of the mature protein that were predicted to form the intrinsically disordered segment (Figure 1A). Additionally, we prepared a synthetic peptide corresponding to the extramembrane N-terminal domain that was predicted to be disordered.Figure 1

Bottom Line: Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes.Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Wilfrid Laurier University, 75 University Avenue West, Waterloo, ON, N2L 3C5, Canada. camp0410@mylaurier.ca.

ABSTRACT

Background: The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane.

Results: We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.

Conclusion: Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

Show MeSH
Related in: MedlinePlus