From the Genetics and Biochemistry Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892
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INTRODUCTION |
Protein translocation across the bacterial inner membrane
(IM)1 and the eukaryotic
endoplasmic reticulum are closely related processes (reviewed in Ref.
1). A highly conserved, membrane-embedded heterotrimer called the
SecYEG complex in bacteria and the Sec61p complex in eukaryotes (2, 3)
forms the core component of the translocation machinery or
"translocon" in both systems. Genetic studies in Escherichia
coli and Saccharomyces cerevisiae have demonstrated the
importance of the SecYEG-Sec61p complex for protein translocation
in vivo (4, 5). The E. coli SecYEG complex and
the peripheral membrane protein SecA, which provides an ATP-driven push, are sufficient to support translocation of preproteins into proteoliposomes in vitro (6). Likewise, the mammalian Sec61p complex can be cross-linked to nascent secretory proteins (2) and is
sufficient to mediate translocation into lipid vesicles (7).
A variety of studies have indicated that in addition to catalyzing
protein translocation, the Sec61p-SecYEG complex facilitates the
insertion of membrane proteins into the endoplasmic reticulum or IM.
Genetic and biochemical studies have demonstrated that the Sec61p
complex plays a central role in the insertion process in both yeast and
mammals (7-9). Several studies using conditional alleles of
secY isolated in screens for protein export mutants have
suggested that the insertion of model inner membrane proteins (IMPs) is
SecY-dependent (10-13). Consistent with these results, depletion of SecE was recently shown to block the insertion of the
maltose transporter MalF (14). However, several other studies that also
used conditional alleles of secY have suggested that the
insertion of some IMPs is SecY-independent (10, 12, 15) or that SecY
dependence is related to the length of cytoplasmic loops (16). Although
the discrepancies in these studies may be due in part to the use of
different assays for IMP insertion, they nevertheless raise the
possibility that some mutant alleles may not affect the transport of
all proteins equally.
Protein insertion into and translocation across a
phospholipid bilayer are two processes that are likely to impose very
different functional requirements on the translocon. The mechanism by
which the SecYEG-Sec61p complex directs both of these processes is
still poorly understood. Available evidence indicates that the
mammalian translocon forms an aqueous channel that permits
translocation of hydrophilic polypeptides (17-19) and also opens
laterally to allow the exit of transmembrane regions into the lipid
bilayer (20, 21). One study indicated that cleavable signal peptides of
secreted proteins and signal-anchor domains of transmembrane proteins
are positioned differently within the eukaryotic translocon, suggesting
that secreted and membrane proteins might be handled differently (22).
In addition, the E. coli translocon may be able to recognize
the longer hydrophobic segments of signal anchors or transmembrane
regions of IMPs and allow them to partition into the lipid bilayer
(23). A recent study suggests that during co-translational targeting,
the mammalian ribosome can detect the presence of an emerging
membrane-spanning domain and use this information to influence the
activity of the translocon (24).
Given the complexity of the tasks performed by the translocon, it is
possible that the passage of secreted proteins and the insertion of
integral membrane proteins are facilitated by distinct regions of the
heterotrimer. Thus, some mutations may preferentially affect the
transport of one of these two classes of proteins. We hypothesized that
one such mutation might be the E. coli secY40 allele, which
was isolated in a screen for secretion mutants (25, 26) based on the
observation that protein export defects up-regulate secA
expression (27, 28). Although the mutation gives rise to a moderate
increase in secA synthesis and a clear cold-sensitive phenotype, no significant defects in maltose-binding protein (MBP) or
OmpA export were observed even after the cells were incubated at the
nonpermissive temperature for prolonged periods (25, 26).
In this study, we tested the possibility that the secY40
mutation selectively affects IMP insertion using a combination of genetic and biochemical experiments. We exploited the observation that
secreted proteins and IMPs are targeted to the IM of E. coli by different pathways. At least some IMPs are targeted by the signal
recognition particle (SRP) (29, 30), a ribonucleoprotein that is
composed of homologs of the mammalian 54-kDa signal sequence binding
protein ("Ffh") and 7SL RNA ("4.5 S RNA") (31, 32). In
contrast, exported proteins are targeted by the chaperone SecB (33) or
by other SRP-independent mechanisms (29). Although SRP is essential for
viability (34, 35), we found that secY40 strains were
hypersensitive to perturbations of the SRP pathway. This result
suggested that the secY40 mutation exacerbates the effect of
SRP mutations by having a similar effect on IMP biogenesis. Direct
examination revealed that the secY40 mutation blocked IMP insertion but, as expected, had little or no effect on the export of
most proteins. These results confirm that secY is required for IMP biogenesis and provide the first evidence that mutations in
secY can have different effects on protein export and IMP
insertion.
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EXPERIMENTAL PROCEDURES |
Reagents, Media, and Bacterial Manipulations--
Antisera
against various proteins were obtained from 5 Prime
3 Prime
(alkaline phosphatase (AP) and
-lactamase (BLA)), New England
Biolabs (MBP), Dr. Jon Beckwith (ribose binding protein (RBP) and
DegP), and Dr. Stephen Pollitt (OmpA). Basic media preparation and
bacterial manipulations were performed using standard methods (36).
Selective media contained 100 µg/ml ampicillin or 40 µg/ml chloramphenicol. The bacterial strains used in this study and their
genotypes are listed in Table I.
Plasmid Construction--
Derivatives of pBR322 and pHDB3 that
overexpress IMPs and derivatives of pACYC and pNU74 containing AP
fusions to IMPs have been described previously (29). For the
experiments described here, a BamHI-SalI
fragment of plasmid pJS310 containing the MtlA 310-AP fusion was cloned
into a derivative of pLG388 (37). The AcrB 576-AP fusion was placed
under control of the lac promoter by transferring a
BlpI-HindIII fragment containing the fusion into
the lacIQ- containing plasmid RB11 (obtained
from Carol Gross). A HindIII-BstEII fragment
from plasmid pHI-1 (38) containing the AP gene was cloned into pWJC12
(39) to create plasmid pAP-1. To obtain plasmid pFFH (wild type), the
BglI site immediately downstream of the ffh
coding sequence was first converted to a BamHI site. A
SalI-BamHI fragment containing the
ffh gene was then cloned into the corresponding sites in
pACYC184. Leucine residues 38 and 39 in Ffh were changed to alanines by
PCR using the "megaprimer" technique (40) to create the ffh
cs101 mutant.
Pulse-chase Labeling and Immunoprecipitation--
Pulse-chase
labeling in M9 medium was performed essentially as described (29)
except that 0.4% maltose was substituted for glucose in experiments in
which MBP export was assayed. For experiments in rich medium, saturated
cultures were diluted into fresh LB to an optical density
(A550) of 0.005. When the cultures reached an
A550 of 0.5 they were labeled for 5 min with 380 µCi/ml Tran35S-label (ICN, specific activity 1490 Ci/mmol). In temperature shift experiments, cultures were grown to an
A550 of 0.4-0.5 at 37 °C and then
transferred to 23 °C for varying lengths of time prior to labeling.
Samples were processed as described (29), and immunoprecipitations were
performed with the following modifications. Trichloroacetic
acid-precipitated samples were solubilized in IP1 (5% SDS, 500 mM Tris-HCl, pH 9.5, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride), heated at 50 °C for
30 min and then diluted with 11.5 volumes of IP2 (2.17% Triton X-100,
163 mM NaCl, 50 mM Tris-HCl, pH 8, 5.5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride).
After binding the antibody-antigen complexes to protein A-agarose, the beads were washed three times with IP3 (0.1% Triton X-100, 0.02% SDS,
150 mM NaCl, 50 mM Tris-HCl, pH 8, 5 mM EDTA) and once with IP4 (150 mM NaCl, 50 mM Tris-HCl, pH 8, 1 mM EDTA).
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RESULTS |
The secY40 Mutation Is Lethal in Strains That Have SRP
Deficiencies--
We reasoned that if the secY40 mutation
inhibits the insertion of IMPs at the permissive temperature, then a
second genetic change that also impairs IMP biogenesis might have a
synergistic effect and lead to a synthetic lethal phenotype. Slight SRP
deficiencies can be tolerated by wild-type E. coli, but a
5-8-fold decrease in the intracellular Ffh concentration becomes
growth-limiting (29), presumably because the biogenesis of IMPs is
reduced below a critical level. Therefore, assessment of the viability
of secY40 strains that have reduced SRP activity should
provide an indication as to whether the mutation impedes IMP insertion.
In support of this notion, we found that the secY39
mutation, which has been shown to inhibit IMP insertion (16), is lethal
in strains carrying SRP deficiencies (data not shown).
To determine whether the secY40 allele affects the viability
of cells that have reduced levels of SRP, we constructed strains HDB84
(secY+) and HDB85 (secY40) in which
expression of ffh is regulated by the trc
promoter. Cells were plated on LB agar containing various amounts of
IPTG and incubated at 37 °C. Previous work demonstrated that the
parent strain (HDB45) produces near normal levels of Ffh in the
presence of 10 µM IPTG (29). HDB84 grew at all IPTG concentrations between 0 and 20 µM, although noticeably
smaller colonies were produced below 2 µM IPTG (Fig.
1A, diamonds). In contrast, HDB85 showed an exquisite sensitivity to the level of Ffh.
Reduction in the IPTG concentration from 20 to 10 µM
resulted in the production of slightly smaller colonies (Fig.
1A, circles); at 5 µM IPTG, a
>50-fold decrease in plating efficiency was observed. Similar results
were obtained using strains in which expression of the gene encoding
4.5 S RNA (ffs) is regulated by the trc promoter. HDB86 (secY+) grew normally at all IPTG
concentrations between 50 and 100 µM (Fig. 1B,
triangles). HDB87 (secY40), however, produced
progressively smaller colonies below 100 µM IPTG, and at
50 µM IPTG the plating efficiency dropped sharply (Fig.
1B, squares). These results show that even a
slight decrease in SRP concentration below the wild-type level inhibits
the growth of secY40 strains and indicate that, even at the
permissive temperature, the secY40 mutation compounds the
effect of perturbing the SRP pathway.

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Fig. 1.
secY40 strains are hypersensitive to
reductions in Ffh and 4.5 S RNA concentration. HDB84
(ffh::kan Ptrc-ffh
secY+; diamonds) and HDB85 (HDB84
secY40; circles) (panel A) and HDB86
(ffs::kan Ptrc-ffs
secY+; triangles) and HDB87 (HDB86
secY40; squares) (panel B) were grown
to saturation in LB containing 20 and 100 µM IPTG,
respectively. Cells were diluted in LB, and approximately 300 colony-forming units were plated on LB agar containing various
concentrations of IPTG and incubated at 37 °C for 14 h.
Relative colony size was scored as follows: 4, colony size
indistinguishable from that observed at highest concentration of
inducer; 3, colony diameter >75% of maximum; 2,
colony diameter between 50 and 75% of maximum; 1, colony
diameter <50% of maximum; 0, colony number reduced
>95%.
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Similar results were obtained when the secY40 mutation was
combined with an Ffh Cs mutation that is likely to reduce substrate binding activity. The ffh cs101 allele contains a mutation
in a highly conserved motif that does not affect protein stability (data not shown) but that has been shown to reduce the signal sequence
binding activity of canine SRP54 (the mammalian SRP 54-kDa protein)
(41). Cells containing the mutation grow at 37 °C or above but do
not grow at 30 °C. To test the ability of cells to tolerate both the
ffh and secY mutations, strains HDB74
(secY+) and HDB78 (secY40), in which
expression of ffh is controlled by the araBAD
promoter, were transformed with a plasmid bearing either the wild-type
ffh gene or ffh cs101. Cells were incubated at
37 °C in the absence of arabinose to repress expression of the
chromosomal ffh gene. HDB78 and HDB74 transformed with pFFH (wild type) grew equally well (Table II). HDB74 transformed with pFFH
(cs101) also grew well, although slightly more slowly than cells transformed with pFFH (wild type). However, HDB78 transformed with pFFH (cs101) did not grow, indicating a synthetic
lethal interaction between the secY40 and ffh
cs101 alleles. Taken together with the results described above,
these data suggest that a modest SRP deficiency and the
secY40 mutation cannot be combined in a single cell because
their effect on IMP insertion is additive.
IMP Insertion Is Impaired in Cells Containing the secY40
Allele--
The results of the synthetic lethality test prompted us to
assess the membrane insertion of several IMPs in secY40
cells directly by using a protease protection assay. In this assay,
protease treatment of spheroplasts releases the AP domain from IMP-AP
fusion proteins that are properly inserted into the IM but not from
those that are retained in the cytoplasm (42). HDB58
(secY+) and CU165 (secY40) were
transformed with plasmids encoding IMP-AP fusions, grown in M9 medium
enriched with amino acids, and radiolabeled. Spheroplasted cells were
treated with protease, and the released AP plus any protected fusion
protein were recovered by immunoprecipitation with an anti-AP
antiserum.
We found that CU165 grown at the permissive temperature (37 °C) had
a small but reproducible defect in the insertion of all IMPs tested.
Small amounts of full-length LctP 426-AP (lactate permease fusion) were
observed in CU165 pulse-labeled for 30 s (Fig.
2A, lane 3).
Significant amounts of full-length LctP 426-AP and AcrB 576-AP
(multidrug efflux pump fusion) also remained in the cytoplasm after a
5-min chase (Fig. 2, A and B, lane 4)
and at later time points (data not shown). A larger amount of
full-length fusion protein was consistently observed after the chase
presumably because the synthesis of the C-terminal AP tag on many
molecules was not completed within the pulse labeling period.
Substantial amounts of protease-protected MalF 350-AP (maltose
transporter fusion) and MtlA 310-AP (mannitol permease fusion) were
observed in pulse-labeled CU165, but less of the intact fusion protein remained after a 5-min chase (Fig. 2, C and D).
Thus, the insertion of these proteins may be delayed rather than
completely blocked by the secY40 mutation. Insertion defects
were also observed for all of these proteins in CU165 after shifting to
23 °C for 90 min, but the magnitude of the defects did not increase
under these growth conditions (data not shown).

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Fig. 2.
IMP insertion is impaired in
secY40 cells grown in minimal medium. HDB58
(secY+, lanes 1 and 2) and
CU165 (secY40, lanes 3 and 4)
transformed with a plasmid expressing LctP 426-AP (A), AcrB
576-AP (B), MalF 350-AP (C), or MtlA 310-AP
(D) were grown to midlog phase at 37 °C in M9-glucose
medium supplemented with amino acids. Insertion of IMP-AP fusions was
assessed by pulse-chase analysis followed by immunoprecipitation with
anti-AP antibodies (see "Experimental Procedures"). The length of
the chase is indicated. The mobilities of the intact fusion proteins
and the protease-resistant AP domain are indicated.
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Consistent with previous reports (25, 26), we found that protein export
was normal in CU165 grown in defined minimal medium. The
SecB-dependent proteins MBP and OmpA, and the
SecB-independent proteins RBP, BLA, and AP were immunoprecipitated from
radiolabeled cells generated in the experiments described above or from
cells transformed with pAP-1, a plasmid that encodes native AP. In each case, a portion of the sample that had not been treated with protease was used (see "Experimental Procedures" and Ref. 29). The
preprotein form of each of these proteins was converted to the mature
form in CU165 as rapidly as in HDB58 (Fig.
3, lanes 1-4), indicating that the secY40 allele did not affect protein export. In
agreement with previous reports (25, 26), incubation at the
nonpermissive temperature (23 °C) also did not reveal a secretion
defect (data not shown). In contrast, the addition of sodium azide, an
inhibitor of SecA function, effectively blocked protein translocation
and led to substantial preprotein accumulation (Fig. 3, lane
5). Hence, under the growth conditions used in these experiments,
the secY40 allele inhibits only IMP insertion.

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Fig. 3.
Protein export is unaffected by the
secY40 mutation in cells grown in minimal medium. BLA,
RBP, AP, OmpA, and MBP were immunoprecipitated from samples generated
in the experiment shown in Fig. 2 that had not been subjected to
proteolysis. AP was immunoprecipitated from cells that had been
transformed with pAP-1. Lanes 1 and 2, HDB58;
lanes 3 and 4, CU165. In lane 5, HDB58
cells were treated with 2 mM sodium azide for 10 min prior
to pulse labeling (HDB58 +N3). Preproteins
(p) and mature proteins (m) are indicated.
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Elevated IMP Insertion Defects Are Observed under Conditions in
Which secY40 Cells Exhibit Strong Growth Defects--
The fact that
the secY40 allele was originally isolated in rich medium
(25, 26) and the observation that IMP insertion defects in cells grown
in minimal medium did not show a clear temperature dependence prompted
us to reexamine the viability of a secY40 strain in
different growth media. MC4100, HDB58, and CU165 were streaked onto LB
agar, M9-glucose agar enriched with amino acids, and M9-glycerol agar
and incubated at 37 or 23 °C. As expected, CU165 did not grow at
23 °C on LB agar (Fig. 4). On enriched
M9-glucose agar, however, CU165 grew at 23 °C, but the colonies had
an abnormal, mucoid appearance. On the poorest medium, M9-glycerol
agar, CU165 grew as well as HDB58 and MC4100 and formed normal colonies
at both 37 and 23 °C. These results suggest that the severity of the
Cs phenotype associated with the secY40 allele correlates
with the rate of cell growth.

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Fig. 4.
The cold-sensitive phenotype of the
secY40 allele depends upon growth conditions. Cells
were streaked from cultures that had just reached stationary phase onto
duplicate agar plates containing LB, M9-glucose supplemented with amino
acids, or M9-glycerol medium. Plates were incubated at 37 °C for
17 h (LB), 24 h (M9 + glucose + AA), or
48 h (M9 + glycerol) and at 23 °C for 36 h
(LB), 48 h (M9 +glucose + AA), or 108 h
(M9 + glycerol).
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To determine whether the secY40 mutation caused a more
substantial IMP insertion block at 23 °C than at 37 °C under
conditions of rapid growth, we developed a pulse labeling method to
analyze the fate of polypeptides synthesized by cells grown in rich
medium. HDB68 and CU165 were transformed with plasmids that expressed IMP-AP fusions, grown to midlog phase in LB, and radiolabeled for 5 min. AP-containing polypeptides were recovered from untreated and
protease-treated spheroplasts by immunoprecipitation as described above. At 37 °C, approximately 22% of the pulse-labeled MalF 350-AP synthesized in CU165 was not inserted into the IM (Fig.
5A, compare lanes 3 and 4). Small or moderate insertion defects were also observed for other IMPs (data not shown). Shifting the incubation temperature to 23 °C, however, increased the magnitude of the insertion defect. After 48 and 114 min at low temperature, 47 and 50%,
respectively, of the pulse-labeled MalF 350-AP was retained in the
cytoplasm (lanes 3 and 4 in Fig. 5, B
and C). In contrast, HDB58 showed no significant
accumulation of MalF 350-AP at 37 °C (Fig. 5A,
lanes 1 and 2) or at 23 °C (Fig. 5,
B and C, lanes 1 and 2).
Thus, the secY40 mutation exerts a greater effect on IMP insertion at low temperature when cells are grown in rich medium.

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Fig. 5.
IMP insertion in secY40 cells
grown in rich medium. HDB58 (lanes 1 and 2)
and CU165 (lanes 3 and 4) transformed with a
plasmid expressing MalF 350-AP were grown in LB medium at 37 °C to
midlog phase. A, a portion of the culture was pulse-labeled
at 37 °C for 5 min as described (see "Experimental Procedures").
The remainder was shifted to 23 °C and incubated for 48 min
(B) or 114 min (C) prior to removing an aliquot
for pulse labeling. Cells were spheroplasted and treated with
(lanes 2 and 3) or without (lanes 1 and 4) proteinase K. Samples were immunoprecipitated with
anti-AP antibody.
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In contrast, the secY40 mutation had little or no effect on
the export of most proteins when cells were grown in LB. Protein export
was assessed by immunoprecipitating OmpA, RBP, BLA, AP, and DegP from
pulse-labeled HDB58 and CU165 transformed with a plasmid expressing an
IMP-AP fusion or with pAP-1. In these experiments, the rate of protein
translocation in the two strains at 37 °C was comparable (Fig.
6, lanes 1 and
2), although the precursor form of several exported proteins
could be detected in CU165 by Western blot (data not shown). Similar
results were obtained at 23 °C (Fig. 6, lanes 3 and
4) except that RBP export was clearly impaired (Fig. 6, RBP,
lane 4). The marked effect on RBP export at low temperature
was unique, however, and we cannot determine from these data whether
the export block was a direct consequence of the mutation or an
indirect effect of the decreased efficiency of IMP insertion.
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Table II
Synthetic lethal interaction between secY40 and ffh mutant
alleles
HDB74 (sec +) or HDB78 (sec 40)
were transformed with a plasmid that expresses either a mutant
ffh allele (pFFH (cs101)) or wild-type ffh (pFFH
(WT)) as a control, plated on LB agar containing 50 µg/ml
chloramphenicol, and incubated at 37 °C for 16 h. Colony
formation was scored as follows: +++, colonies indistinguishable from
control; ++, colony diameter between 50 and 100% of control; +, colony
diameter <50% of control; , colony number reduced >95%.
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Fig. 6.
Protein export in secY40 cells
grown in rich medium. Proteins were immunoprecipitated from HDB58
(lanes 1 and 3) and CU165 (lanes 2 and
4) transformed with a plasmid expressing AP (OmpA, AP, DegP)
or an IMP-AP fusion (RBP, BLA). Cells were grown at 37 °C in LB to
midlog phase. One portion of the culture was pulse-labeled at 37 °C
(lanes 1 and 2), and another portion was
incubated at 23 °C for 48 min prior to pulse labeling (lanes
3 and 4). In lane 5, HDB58 cells were
treated with 2 mM sodium azide for 10 min prior to pulse
labeling.
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DISCUSSION |
In this report, we show that the secY40 mutation
selectively blocks the insertion of IMPs. Initially, we found that
combining the secY40 mutation with SRP deficiencies produced
a synergistic effect and reduced cell viability. The genetic data
suggested that the secY40 mutation blocks the same
biological process as ffh and ffs mutations.
Consistent with these results, direct examination of the insertion of
several individual IMPs revealed that the secY40 mutation
has a broad effect on IMP biogenesis. Moderate IMP insertion defects
were observed in cells grown at 37 °C in both rich and minimal
media. Shifting cells grown in LB to 23 °C led to more severe IMP
insertion defects. In contrast, no defects in the export of
SecB-dependent proteins (which had been examined previously) or several SecB-independent proteins could be detected in
cells grown in minimal medium. The export of most proteins was not
notably affected by growth in rich medium either. Taken together, these
results suggest that the marked cold sensitivity of secY40
strains grown in rich medium is attributable to an increased impairment
of IMP insertion defects at low temperature.
The data presented here help to clarify two issues regarding SecY
function. First, the observation that the insertion of several unrelated IMPs was blocked by the secY40 mutation supports
the view that the SecYEG translocon is required for IMP biogenesis. The
observation that the mutation affects the insertion of both MalF, which
has a long periplasmic loop, and MtlA, which has only short periplasmic
loops, also suggests that the translocon facilitates the insertion of a
broader range of IMPs than has been previously proposed (16). Second,
the finding that the secY40 mutation inhibits IMP insertion
links the mutation to the protein transport function of SecY and argues
against the proposal that it affects a postulated second function of
SecY (25). Our data do not fully explain, however, why the
secY40 mutation was isolated in a screen for mutations that
increase secA expression. One possibility is that an
inhibition of IMP insertion, like a protein export block, leads to an
increase in SecA synthesis. This possibility is consistent with several
studies that indicate that SecA is required for the insertion of at
least some IMPs (10, 14, 16, 43). Alternatively, the slight protein
export defects observed in secY40 strains when cells are
grown in rich medium may be sufficient to cause a moderate up-regulation of secA expression. This explanation is
consistent with the observation that the secY40 mutation
increases secA expression to a much smaller degree than
secY alleles that produce strong effects on protein export
(26).
An important point that arises from this study is that it cannot be
assumed that translocon mutations have equal effects on the transport
of all proteins. For this reason, the use of a single sec
allele to test the "Sec dependence" of transport of a given protein
may yield misleading results. Another implication of this study is that
transport defects may be sensitive to growth conditions. The
observation that both the phenotype and the biochemical defects associated with the secY40 allele varied in different media
demonstrates that it can be misleading to extrapolate from one growth
condition to another. Slowing the growth rate of secY40
cells by culturing in minimal medium may suppress the cold-sensitive
phenotype by reducing the burden of polypeptides that the translocon
must handle in a given time period. This notion is consistent with the
observation that the secY40 Cs phenotype can be suppressed
by simply overproducing a cytoplasmic protein (44), which presumably
reduces the rate of synthesis of IMP proteins. Sensitivity to
biosynthetic rates may be a general property of sec
mutations, since the growth defects of several Cs alleles of
secY, secE, secD, and secF
can all be suppressed by overproduction of cytoplasmic proteins
(44).
The most intriguing implication of this study is that translocon
mutations can have distinct effects on protein secretion and membrane
protein insertion. Our results suggest that different regions of
SecY/Sec61p may be specialized to facilitate two fundamentally distinct
processes, the translocation of largely hydrophilic proteins and the
membrane insertion of more hydrophobic IMPs. Although a variety of
export-defective alleles of secY have been obtained based on
the up-regulation of secA expression, the mutations tend to
cluster in specific regions of the protein (26). Indeed, no mutations
have been isolated in some highly conserved regions that might
conceivably participate in IMP insertion. For these reasons, it should
be interesting to develop additional screening methods to isolate new
secY alleles that impair the transport of specific classes
of proteins. Characterization of these alleles as well as further
analysis of existing sec mutations should provide a great
deal of insight into the mechanism by which the translocon performs a
complex set of tasks.
We thank Carol Gross, Koreaki Ito, and Jon
Beckwith for gifts of plasmids, strains, and antibodies and Gisela
Storz for critical reading of the manuscript.