(Received for publication, October 2, 1996, and in revised form, November 28, 1996)
From the Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel
In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor. In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally. Unexpectedly, despite this variance, E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery. In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E. coli. The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E. coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions.
Considerable effort has been devoted to studying how soluble proteins are selectively targeted and translocated across biological membranes. In contradistinction, similar questions dealing with the biogenesis of complex membrane proteins in Escherichia coli remained poorly understood. The involvement of cell factors in the biosynthetic pathway of membrane proteins, unlike translocated proteins, is also unknown, and recent studies on the role of the Sec machinery in this process are contradicting (1, 2). Another unresolved question is how prokaryotic membrane proteins are targeted to the cytoplasmic membrane, and what are the cellular mediators of this process. In mammalian cells, targeting of many membrane and secretory proteins is mediated by the signal recognition particle (SRP)1 machinery (3, 4). In E. coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally (5) with the aid of chaperones (6). However, it has been shown that E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery (7, 8). The function of these proteins in translocation has remained controversial, since their depletion induces only relatively small translocation defects for some secretory proteins (9, 10). In addition, their role in biogenesis of membrane proteins is not yet clear. In this study we present evidence obtained in vivo that FtsY, the E. coli SRP-receptor homologue, is essential for expression of membrane proteins.
To study the possibility that the E. coli SRP-receptor
homologue, FtsY, is required for biosynthesis of polytopic membrane proteins, we used the E. coli strain
N4156::pAra14-FtsY (10) which contains a chromosomal copy of
the essential ftsY gene under the control of the tight
araB promoter (instead of the native ftsY) and
therefore requires arabinose for growth (10). The effect of arabinose
depletion on cell growth was analyzed at various times after the
cultures were transferred to arabinose-free medium. Arabinose-depleted
cells begin to show a growth defect relative to wild-type cells after
4 h (Fig. 1A). The expression of FtsY in
cells grown in the absence of arabinose is markedly reduced after
2 h and essentially ceases after 4 h (Fig. 1B).
The level of the cytoplasmic protein
-galactosidase is similar in
induced or uninduced cells (Fig. 1, C and G),
while the amount of the membrane protein lac permease is
dramatically decreased in the absence of arabinose, both in membrane
preparations and in whole cell extracts (Fig. 1, D and
G). Since
-galactosidase and lac permease are
expressed from the same operon, the difference in their expression
pattern must reflect post-transcriptional, FtsY-related events. The
pattern of
-lactamase expression is similar to that of
-galactosidase (Fig. 1, E and G), being only
slightly affected in FtsY-depleted cells, while the accumulation of the
chromosomally encoded protein, SecY, is markedly inhibited (Fig. 1,
F and G). SecY is a multispanning membrane
protein that functions as a component of the translocation machinery
(reviewed in Ref. 11). As shown in Fig. 1, E and
H, and in agreement with previous studies (10), the
efficiency of the translocation of
-lactamase decreases during FtsY
depletion, as judged by the accumulation of pre-
-lactamase. These
results show that the synthesis of the two membrane proteins tested
(SecY and lac permease) is greatly reduced in FtsY-depleted cells, whereas that of a secretory protein (
-lactamase) is only slightly affected and that of a cytoplasmic protein (
-galactosidase) not at all.
Effect of FtsY depletion. A,
growth curves of wild-type and FtsY-depleted cells (an average of 3 independent experiments). B, Western blot analysis with
anti-FtsY antibodies of cell extracts prepared from wild-type and
FtsY-depleted cells taken at the indicated times after arabinose
depletion. C, Western blot analysis with anti--galactosidase antibodies, of extracts prepared from
isopropyl-1-thio-
-D-galactopyranoside-induced cells
taken at the indicated times after arabinose depletion. D,
Western blot analysis with anti-lac permease antibodies of membranes and whole cell extracts prepared from
isopropyl-1-thio-
-D-galactopyranoside-induced cells
taken at the indicated times after arabinose depletion. E,
Western blot analysis with anti-
-lactamase antibodies of extracts prepared from arabinose-induced or uninduced FtsY-depleted cells taken
at the indicated times after arabinose depletion. F, Western blot analysis with anti-SecY antibodies of membranes prepared from
arabinose-induced or uninduced FtsY-depleted cells taken at the
indicated times after arabinose depletion. G, effect of FtsY
depletion on the relative expression of
-galactosidase, lac permease, SecY, and the sum of both forms of
-lactamase (precursor + mature). The results of at least 2 independent experiments as those presented in C,
D (left), E, and F were
averaged and manipulated as follows. The relative expression of the
indicated protein in FtsY-depleted cells grown without arabinose is
presented as percentage of its amount in wild-type cells (for
-galactosidase and lac permease) or FtsY-depleted cells
grown with arabinose (for SecY and
-lactamase), at the indicated
times after arabinose depletion. H, efficiency of
-lactamase translocation. The amount of the mature form of
-lactamase at the indicated times is presented as percentage of the
sum of the precursor and the mature forms at the same time points. The
experiments shown here were performed as follows. Wild-type (N4156) or
FtsY-depleted (N4156::pAra14-FtsY
) cells were grown in YT
broth overnight with arabinose (0.2%), washed once in YT broth, and
resuspended in YT (to A600 = 0.01) with or
without arabinose as indicated. Cell extracts were prepared by
sonication in buffer A (50 mM Tris-HCl, pH 8, 0.5 M NaCl, 1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride). Membranes were prepared by sonication
in buffer A supplemented with 50 mM NaOH, followed by low
speed centrifugation (removal of cell debris), and the membranes were
collected by ultracentrifugation (45 min, 200,000 × g). Proteins were solubilized in SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis. The amounts of
proteins loaded in each lane are: B and C, 5 µg; D, left, 5 µg; D,
right, 25 µg; E, 40 µg; and F, 15 µg. Polyclonal anti-FtsY antibodies (B) were kindly
provided by Joen Luirink (Institute of Molecular Biological Sciences,
Amsterdam). Monoclonal anti-
-galactosidase antibodies (C)
were obtained from Boehringer Mannheim, and monospecific anti-lac permease antibodies (D) were kindly provided by H. Ronald Kaback (UCLA). Polyclonal anti-
-lactamase antibodies
(E) were from 5 Prime
3 Prime, and polyclonal anti-SecY
antibodies (F) were kindly provided by Arnold J. Driessen
(University of Groningen). Quantitation of the immunoreactive bands was
done by densitometry, and the data presented represent the averages of
at least three independent experiments.
A possible explanation for the results is that the translation of
membrane proteins is arrested in the absence of FtsY, mimicking the
translation arrest observed in the mammalian targeting system (12-14).
Such an arrest would lead to a deficient number of functional translocation complexes in the membrane and, consequently, to the
accumulation of pre-proteins, especially those with lower affinities to
the translocation site. Provided that FtsY depletion causes a
translational arrest in the case of membrane proteins, then reinduction
of FtsY is expected to release this arrest. To test this suggestion, we
performed an experiment in which arabinose was added to FtsY-depleted
cells. As shown in Fig. 2, addition of arabinose
restores both the cell growth (Fig. 2A) and the expression of FtsY (Fig. 2, B and C) after 1 h.
Similarly, rapid restoration of expression of the membrane proteins
lac permease (Fig. 2, D and E) and
SecY (Fig. 2, F and G) is apparent. However, the
resumption of -lactamase processing occurs only 2 h later (Fig.
2, H and I). Interestingly, during the 2-h lag
after addition of arabinose, the processing of pre-
-lactamase is
significantly inhibited compared to uninduced cells, in a highly
reproducible manner (Fig. 2I). A speculative explanation
is that immediately after induction of FtsY, many newly synthesized
membrane proteins in addition to pre-proteins which have been
accumulated during the FtsY depletion period, would compete, some of
them with better affinities than
-lactamase, for the limited number
of translocation sites. In any case, the translocation defect seen for
-lactamase may be caused indirectly via the depletion of SecY and
other components of the translocation complex.
Our results reveal that the biogenesis of membrane proteins such as lac permease and SecY is strongly dependent on FtsY. Although the mechanism of targeting of membrane proteins in bacteria is still an open question, the involvement of FtsY supports a co-translational pathway, analogous to the mammalian SRP-mediated pathway. Previous observations that FtsY is located both in the cytoplasm and in association with the inner membrane (10, 15) indicate that it may function not only in the last stage of membrane protein targeting, but also in directing the translation complex to the membrane, possibly via an interaction with the prokaryotic SRP-like complex (16). The present assignment of FtsY as an important participant in the biosynthetic pathway of membrane proteins provides a reasonable explanation for the observations that FtsY is essential for growth (10, 17).
We thank Dr. A. S. Girshovich for his helpful
suggestions during the preparation of the manuscript, Dr. T. A. Rapoport for critically reading the manuscript, and the members of the
Bibi laboratory and Dr. S. Michaeli for their critical evaluation of this work. We are grateful to Dr. Joen Luirink for supplying E. coli strains N4156 and N4156::pAra14-FtsY and anti-FtsY
antibodies and Drs. A. J. M. Driessen and W. Wickner for the anti-SecY
antibodies.