(Received for publication, August 21, 1995; and in revised form, October 6, 1995)
From the
We have shown previously that the 100-residue-long periplasmic N-terminal tail of the Escherichia coli inner membrane protein ProW can be translocated across the inner membrane in a sec-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G.(1994) EMBO J. 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation. Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions. We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues. Thus, sec-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.
The insertion of proteins into the inner membrane of Escherichia coli has been studied intensely over the past few years, and it is now generally accepted that hydrophobic segments drive insertion and end up spanning the membrane, whereas the orientation of the transmembrane segments is controlled by flanking positively charged residues: the ``positive inside'' rule(1) . The details of the insertion mechanism are less clearly understood, however. In general, it seems that long periplasmic segments are translocated across the inner membrane by the so-called sec machinery also used by secretory proteins, whereas short periplasmic segments in most cases do not seem to require a fully functional sec machinery for translocation(2) .
We
recently described an interesting exception to this correlation between
the length of a translocated domain and its sec-dependence;
the 100-residue-long periplasmic N-terminal tail (N-tail) ()of the ProW protein can be efficiently translocated across
the inner membrane under conditions where the function of SecB, SecA,
or SecY is severely compromised(3) . Insertion of three
positively charged arginines near the C-terminal end of the ProW N-tail
blocks its sec-independent translocation, whereas insertion of
three negatively charged aspartic acid residues has no effect.
Dissipation of the electrochemical potential across the inner membrane
also blocks translocation of the N-tail, suggesting an electrophoretic
component in the translocation mechanism.
We now report that insertion of a single arginine residue near the C-terminal end of the N-tail severely affects translocation, whereas insertion of two arginines blocks translocation almost completely irrespective of position. Further, we show that all of the ProW protein downstream of the most N-terminal transmembrane segment can be deleted with little effect on N-tail translocation, provided that a few positively charged residues are placed at the C-terminal end of the remaining transmembrane segment. To our knowledge, this is the first instance where the translocation of a large polar domain across the inner membrane has been shown to be induced by a translocation signal located at the extreme C terminus of the protein.
For all samples, the fraction of background-corrected intensities in the protease-protected fragment band b (protease-treated cells; Fig. 1B, arrow) relative to bands a and b (where band a is the full-length protein in the protease-treated sample) was calculated.
On-line formulae not verified for accuracy
Figure 1:
A, topologies of ProW and the
ProW-Lep(P2) fusion (I) as well as the
ProW
/MKKK (II) and ProW
/MM
(III) mutants. Wild type ProW has seven transmembrane segments (black and gray bars), three of which are retained in
the ProW
-Lep(P2) fusion (black bars).
The positions of the arginine insertions are indicated. In the
ProW
-Lep(P2) fusion, the periplasmically exposed
ProW N-tail can be removed by protease treatment of spheroplasts,
leaving a protease-resistant fragment composed of the three ProW
transmembrane segments and the Lep(P2) domain. B, topological
mapping of the ProW
(3R)-Lep(P2) construct. Cells
expressing the fusion protein were labeled by
[
S]methionine for 1 min, converted to
spheroplasts, treated with proteinase K, and processed for
immunoprecipitation with Lep antiserum. Band a is the intact
fusion protein, and band b is the protease-resistant fragment
resulting from proteolytic removal of the periplasmically exposed ProW
N-tail(3) . Azide was added to the cells to block the function
of SecA (lanes 3 and 4), and CCCP was added to
dissipate the electrochemical membrane potential (lanes 5 and 6).
All quantitations were carried out on a Fuji BAS 1000 Image reader using the MacBAS (2.1) software. For the experiments reported in Fig. 4, the translocation efficiency was determined by normalizing the intensity in the ProW band by the intensity in the band corresponding to the cytoplasmic control AraB and then comparing the amount of ProW immunoprecipitated from samples either treated with proteinase K or left untreated.
Figure 4:
A,
topological analysis of the ProW/MKKK mutant.
Note that the non-translocated precursor form of the outer membrane
control protein OmpA accumulates when cells are treated with azide or
CCCP prior to pulse labeling (lanes 3-6). B,
topological analysis of the ProW
MM mutant. After
pulse labeling, ProW, OmpA (an outer membrane protein that becomes
protease-sensitive only upon disruption of the outer membrane), and
AraB (a cytoplasmic protein) were immunoprecipitated by their
respective antisera and analyzed by SDS-polyacrylamide gel
electrophoresis. p, pro-OmpA; m, mature
OmpA.
To test the
dependence on the electrochemical membrane potential, cells were
treated as in a normal protease protection experiment except that CCCP
was added to a final concentration of 50 µM 45 s before
the addition of
[S]methionine(8, 9) .
Somewhat atypically,
the first cytoplasmic loop in ProW does not contain any positively
charged residues, and we reasoned that some of the N-tail mutants
discussed below might give rise to an ``inverted'' topology
with the N-tail in the cytoplasm and the first loop in the periplasm.
Because this would complicate the interpretation of the results, three
positively charged arginine residues were inserted between positions
119 and 120 in the first cytoplasmic loop (mutant
ProW(3R)-Lep(P2)). As shown in Fig. 1B, the N-tail is translocated in this mutant as
judged by its sensitivity to proteinase K added to intact spheroplasts
(92% translocation; lanes 1 and 2), and translocation
is not blocked by sodium azide (lanes 3 and 4),
suggesting that SecA is not required(10) . As for the original
ProW
-Lep(P2) fusion(3) , dissipation of
the membrane electrochemical potential by the protonophore CCCP
completely blocks translocation, leaving the entire fusion protein
resistant to the externally added protease (lanes 5 and 6). We thus used the ProW
(3R)Lep(P2)
mutant in all subsequent experiments.
Figure 2:
Topological mapping of the
ProW(3R,
5-25)-Lep(P2) mutant. See Fig. 1for details.
As
shown in Fig. 3A, a single arginine had a very strong
effect when inserted between positions 99 and 100 (5% translocation; lanes 9 and 10), a milder effect when inserted
between positions 32 and 33 and positions 82 and 83 (30%
translocation; lanes 3 and 4 and lanes 7 and 8), and was well tolerated when inserted between positions 4
and 5 and positions 65 and 66 (80-90% translocation; lanes 1 and 2 and lanes 5 and 6). Two arginines
blocked translocation almost completely in all positions, again with
the exception of positions 65 and 66 where 30% translocation was
observed. For unknown reasons, we failed to express a mutant with two
arginines inserted in position 5 despite repeated attempts.
Figure 3: A, topological mappings of the 1R and 2R series of mutants. See Fig. 1for details. B, quantitation of the results shown in A. The efficiency of N-tail translocation (defined as the ratio between the intensities of band b and bands a and b in the protease-treated samples) for the different 1R and 2R mutants is shown.
We conclude that translocation is particularly sensitive to the introduction of positively charged residues near the C-terminal region of the N-tail next to the first transmembrane segment. Nevertheless, two arginines introduced more than 60 residues away from the first transmembrane segment also block translocation almost completely.
Two ProW truncation mutants with stop
codons placed in position 120 directly after the first transmembrane
segment (residues 100-118) were made to test this possibility:
one ending IAWQMM and the other ending
IAWQ
MKKK. The two mutants differ by the presence of three
extra positively charged lysines in the latter; methinonies were
included to facilitate detection by pulse labeling with
[
S]methionine.
The topological analysis of
the two mutants is presented in Fig. 4. The N-tail of the
ProW/MKKK mutant is partially translocated
(65-70%) both in the absence (Fig. 4A, lanes
1 and 2) and presence (Fig. 4A, lanes
3 and 4) of sodium azide, and its translocation is thus
independent of the SecA protein. Dissipation of the membrane
electrochemical potential by CCCP prevents translocation (Fig. 4A, lanes 5 and 6). Note that
translocation of the sec-dependent outer membrane protein OmpA
is largely blocked by both azide and CCCP, resulting in accumulation of
the precursor form pro-OmpA inside the cell.
In contrast, the
ProW/MM mutant lacking C-terminal positively
charged residues is resistant to protease digestion under all
conditions (Fig. 4B), demonstrating that the N-tail is
not translocated across the inner membrane. Possibly, the C-terminal
end of the hydrophobic segment is translocated in this case, but this
cannot be determined with the present protease assay.
We conclude
that the hydrophobic segment located at the extreme C terminus of the
ProW truncation mutant can act as a signal for
the SecA-independent translocation of the N-tail and that its
orientation can be determined by flanking positively charged residues.
Periplasmically exposed N-tails in E. coli inner membrane proteins contain few positively charged residues and appear to be translocated by a sec-independent mechanism irrespective of length(3, 11) . This is in contrast to long internal periplasmic loops and C-terminal tails for which translocation in most cases is sec-dependent (12) and which can contain high numbers of positively charged residues(1, 13) .
In this paper, we show that the insertion of as little as a single arginine into the ProW N-tail can dramatically affect the efficiency of translocation, whereas two arginines fully block translocation (Fig. 3). The effects on translocation vary with the position of the arginine(s), but none of the double-arginine mutations tested is completely without effect. This suggests that the entire N-tail is critical for translocation, although our data indicate that its C-terminal extremity is more important than its central and N-terminal parts. Nevertheless, deletions near the N and C termini of the N-tail have no effect on translocation ( Fig. 2and (3) ). Thus, the simple rule that positively charged residues are not tolerated in the N-tail irrespective of position holds remarkably well.
The ease
with which the wild type N-tail can be translocated is even more
dramatically illustrated by the finding that a truncation mutant
composed only of the first 119 residues of ProW (i.e. the
N-tail and the first transmembrane segment) followed by a methionine
and three lysine inserts into the inner membrane with the N-tail
exposed to the periplasm (Fig. 4). To our knowledge, this is the
first demonstration that a hydrophobic segment located at the extreme C
terminus of a protein can serve as a translocation signal for a large
N-terminal domain. Previously, this has only been shown to be possible
for small proteins such as the coat protein of phage Pf3(14) ,
which is composed of a single transmembrane segment flanked by 18
N-terminal and 8 C-terminal residues. As expected from the positive
inside rule(1) , translocation of the N-tail in the
ProW truncation mutant is only observed when the
hydrophobic segment is flanked on its C-terminal side by positively
charged residues. Further, translocation of the N-tail is independent
of the sec-machinery, as in all other ProW constructs tested
to date.
Because the translocation signal is located C-terminally to the N-tail, it seems reasonable to assume that the C-terminal end of the N-tail is translocated across the membrane at an early stage, in keeping with the ``helical hairpin'' hypothesis(15) . The position-dependent effects on translocation in the 1R series of mutants analyzed here (Fig. 3) also suggest that the C-terminal end of the N-tail is more critical for translocation than other parts. Previous studies have shown that a segment encompassing the first 20 residues on the C-terminal side of a normal signal peptide in a sec-dependent protein is critical for translocation and highly sensitive to the introduction of positively charged residues(15) , suggesting that the formation of a transmembrane helical hairpin may be an early step in both sec-dependent and sec-independent translocation mechanisms.
Interestingly, an
insertion of two arginines more than 60 residues away from the first
transmembrane segment suffices to block export of the N-tail. It would
seem that the initial formation of a helical hairpin comprising the
first transmembrane segment and the preceding 20 residues should
not be influenced by this mutation; rather, it may be that the two
basic residues prevent the ultimate translocation of the N-terminal end
of the N-tail and force the chain to ``slip back'' through
the membrane(16) .