COMMUNICATION:
FtsY, the Prokaryotic Signal Recognition Particle Receptor Homologue, Is Essential for Biogenesis of Membrane Proteins*

(Received for publication, October 2, 1996, and in revised form, November 28, 1996)

Andrei Seluanov and Eitan Bibi Dagger

From the Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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.


INTRODUCTION

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 beta -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 beta -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 beta -lactamase expression is similar to that of beta -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 beta -lactamase decreases during FtsY depletion, as judged by the accumulation of pre-beta -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 (beta -lactamase) is only slightly affected and that of a cytoplasmic protein (beta -galactosidase) not at all.


Fig. 1.

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-beta -galactosidase antibodies, of extracts prepared from isopropyl-1-thio-beta -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-beta -D-galactopyranoside-induced cells taken at the indicated times after arabinose depletion. E, Western blot analysis with anti-beta -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 beta -galactosidase, lac permease, SecY, and the sum of both forms of beta -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 beta -galactosidase and lac permease) or FtsY-depleted cells grown with arabinose (for SecY and beta -lactamase), at the indicated times after arabinose depletion. H, efficiency of beta -lactamase translocation. The amount of the mature form of beta -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-beta -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-beta -lactamase antibodies (E) were from 5 Prime right-arrow 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.


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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 beta -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-beta -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 beta -lactamase, for the limited number of translocation sites. In any case, the translocation defect seen for beta -lactamase may be caused indirectly via the depletion of SecY and other components of the translocation complex.


Fig. 2. Restoration effects of arabinose addition to FtsY-depleted cells. E. coli N4156::pAra14-FtsY' cells were grown in YT broth overnight with arabinose, washed once in YT, resuspended in YT (to A600 = 0.01) without arabinose. After 4 h of growth, the culture was divided, and arabinose (0.02%) was added to one half (indicated by an arrow). A, growth curves of N4156::pAra14-FtsY' cells with and without addition of arabinose. B, D, F, and H, samples withdrawn at the indicated time from both cultures were treated as described in the legend to Fig. 1 and subjected to Western blotting with the specific antibodies (anti-FtsY, anti-lac permease, anti-SecY, and anti-beta -lactamase, respectively). C, E, G, and I, quantitative analyses of experiments such as those shown in B, D, F, and H, respectively. Quantitation of the immunoreactive bands was done by densitometry. The data presented in C (-arabinose), E, G, and I represent the averages of at least three independent experiments, and the standard error for each point was within 10%. The standard error for each point in C (+arabinose) was within 15%.
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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).


FOOTNOTES

*   The work was supported by the Leo and Julia Forchheimer Center for Molecular Genetics, Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Incumbent of Dr. Samuel O. Freedman Career Development Chair in the Life Sciences. To whom correspondence should be addressed. Tel.: 972-8-934-3464; Fax: 972-8-934-4118; E-mail: BCBIBI@WEIZMANN. WEIZMANN.AC.IL.
1    The abbreviation used is: SRP, signal recognition peptide.

Acknowledgments

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.


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©1997 by The American Society for Biochemistry and Molecular Biology, Inc.