Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

Matthew P. DeLisa; Danielle Tullman; George Georgiou

doi:10.1073/pnas.0937838100

New Research In

Edited by Jonathan Beckwith, Harvard Medical School, Boston, MA, and approved March 7, 2003 (received for review December 23, 2002)

Article Figures & SI

Figures

Tables

Download figure

Open in new tab

Download powerpoint

Figure 1
AP folding and export in different strain backgrounds. Ox and red refer to oxidizing and reducing redox potentials in the specified subcellular compartment. In C:ox (DR473) cells, AP is able to fold in the cytoplasm and thus can serve as a substrate for the Tat pathway. The localization of AP is not impaired in the C:ox/P:red strain DRA (DR473 dsbA). Deletion of tatC (or tatB) in the C:ox/P:ox strain blocks AP export.
Download figure

Open in new tab

Download powerpoint

Figure 2
Subcellular localization of ssFdnG-AP. Immunoblotting of periplasmic (A) and cytoplasmic (B) fractions from cells expressing ssFdnG-AP. Samples were normalized on the basis of the amount of total cell protein and resolved on SDS-12% polyacrylamide gels. GroEL was used as a fractionation marker by probing with anti-GroEL serum. (C) AP activity for the same periplasmic (filled bars) and cytoplasmic (open bars) fractions as in A and B. (D) Trypsin sensitivity analysis of periplasmic fractions collected from C:ox/P:ox and C:ox/P:red cells. Samples were separated on SDS-4–20% polyacrylamide gels and probed with anti-AP and anti-OmpA serum.
Download figure

Open in new tab

Download powerpoint

Figure 3
Tat export of a scFv. Detection of scFv by ELISA in the periplasmic and cytoplasmic fractions. Periplasmic (filled bars) and cytoplasmic (open bars) samples were serially diluted and started from the same amount of total protein. Data reported are from a 4-fold dilution and are the average of two independent experiments.
Download figure

Open in new tab

Download powerpoint

Figure 4
Tat export of an antidigoxin antibody fragment (F_AB). (A) Tat export of F_AB antibodies. (B) Western blotting of periplasmic (lanes 1–3) and cytoplasmic (lanes 4–6) fractions collected from cells coexpressing TorA-Fab fusion and ΔssDsbC. Strains used were (1, 4) C:ox/P:ox; (2, 5) C:ox/P:ox/tatC, and (3, 6) C:red/P:ox. All lanes were loaded with the same amount of total protein and anti-mouse IgG F(ab′)₂ antibody was used to detect the F_AB light chain. GroEL was used as a fractionation marker for the spheroplast fractions. (C) ELISA of periplasmic (a, c, and e) and cytoplasmic (b, d, and f) fractions collected from C:red/P:ox (a and b), C:ox/P:ox/tatC (c and d), and C:ox/P:ox (e and f) cells. (D) Flow cytometric analysis of C:red/P:ox cells (Upper) and C:ox/P:ox cells (Lower) cells expressing TorA-Fab and ΔssDsbC and labeled with FITC-digoxin.

Tables

Figures

View popup

Table 1

Bacterial strains and plasmids used in this study

E. coli strain or plasmid	Relevant phenotype or features	Source
DHB4	MC1000 phoR Δ(phoA) PvuII Δ(malF)3 F′[lacI^qZYA pro]	Laboratory stock
DR473	DHB4 ΔtrxB gor552 Tn10Tet ahpC^* Tn10Cm (araC P_ara-trxB)	J. Beckwith
FA113	DHB4 trxB gor552 Tn10tet^rahpC^*	J. Beckwith
DHBA, DRA	DHB4 dsbA∷kan, DR473 dsbA∷kan	This work
DRB	DR473 tatB∷kan	This work
DRC	DR473 tatC∷spec	This work
pTrc99A	trc promoter, ColE1 ori, amp^r	Amersham Biosciences
pAID135	Δ(2–22)AP	Ref. 20
pTorA-AP	ssTorA-Δ(1–22)AP cloned in pTrc99A	This work
pKK-AP	as pTorA-AP with R11K;R12K mutation in ssTorA	This work
pFdnG-AP	ssFdnG-Δ(1–22)-AP in pTrc99A	This work
pTrc99-Fab		Ref. 22
pssTorA-Fab	ssTorA-V_H-C_H1 and V₁-C₁ dicistronic operon in Trc99	This work
pKK-Fab	as above with ssTorA(R11K;R12K)	This work
pBAD-ΔssdsbC		Ref. 22

View popup

Table 2

Periplasmic alkaline phosphatase activity in cells expressing Tat leader peptide-AP fusions*

FdnG	`MDVSRRQFFKICAGGMAGTTVAALGFAPKQALAQ`
FdoG	`MQVSRRQFFKICAGGMAGTTAAALGFAPSVALAE`
HyaA	`MNNEETFYQAMRRQGVTRRSFLKYCSLAATSLGLGAGMAPKIAWAL`
TorA	`MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAA`
DmsA	`MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHAV`
SufI	`MSLSRRQFIQASGIALCAGAVPLKASAA`
YacK	`MQRRDFLKYSVALGVASALPLWSRAVFAA`
YcbK	`MDKFDANRRKLLALGGVALGAAILPTPAFAT`

Leader peptide	Periplasmic AP activity
Leader peptide	DHA C:red/P:red	DHB4 C:red/P:ox	DR473 C:ox/P:ox	DRA C:ox/P:red	DRB (DR473 tatB∷kan) C:ox/P:ox	DRC (DR473 tatC∷spec) C:ox/P:ox
Δ2–20^†	43	56 (52)	69 (7)	57 (8)	56 (8)	47 (9)
FdnG^‡	32	45 (48)	420 (42)	371 (38)	30 (5)	6 (2)
FdoG	55	62 (54)	446 (53)	385 (50)	17 (2)	11 (2)
HyaA^‡	17	28 (46)	187 (61)	185 (59)	65 (23)	20 (4)
TorA^‡	42	51 (51)	437 (26)	403 (28)	38 (3)	17 (1)
DmsA^‡	35	123 (>90)	280 (23)	175 (14)	112 (12)	74 (9)
SufI^‡	75	323 (>90)	459 (30)	381 (19)	263 (15)	308 (18)
YacK^‡	25	102 (>90)	153 (18)	135 (12)	185 (19)	80 (9)
YcbK	27	95 (>90)	178 (22)	137 (13)	170 (18)	81 (10)

All signal sequences were fused in frame to E. coli AP(Δ1–22), and the fusions were inserted into vector pTrc99A (see Experimental Procedures).
↵* Cells were treated with 100 mM iodoacetamide immediately after harvesting to prevent formation of disulfide bonds during subsequent sampling processing. AP activity was calculated as the amount of p-nitrophenyl phosphate hydrolyzed (in micromoles) per minute at 25°C and pH 8.0. Reported values for AP activity are the average of three separate measurements from two independent experiments (n = 6). Standard error is <10% for all reported data. Values in parentheses indicate the percentage of the total enzymatic activity in the periplasmic fraction.
↵† AP with a Δ2–20 in the native leader peptide.
↵‡ Leader peptide carries a c-region positive charge.