Characterization of the consequences of YidC depletion on the inner membrane proteome of E. coli using 2D blue native/SDS-PAGE

In the bacterium Escherichia coli, the essential inner membrane protein (IMP) YidC assists in the biogenesis of IMPs and IMP complexes. Our current ideas about the function of YidC are based on targeted approaches using only a handful of model IMPs. Proteome-wide approaches are required to further our understanding of the significance of YidC and to find new YidC substrates. Here, using two-dimensional blue native/SDS-PAGE methodology that is suitable for comparative analysis, we have characterized the consequences of YidC depletion for the steady-state levels and oligomeric state of the constituents of the inner membrane proteome. Our analysis showed that (i) YidC depletion reduces the levels of a variety of complexes without changing their composition, (ii) the levels of IMPs containing only soluble domains smaller than 100 amino acids are likely to be reduced upon YidC depletion, whereas the levels of IMPs with at least one soluble domain larger than 100 amino acids do not, and (iii) the levels of a number of proteins with established or putative chaperone activity (HflC, HflK, PpiD, OppA, GroEL and DnaK) are strongly increased in the inner membrane fraction upon YidC depletion. In the absence of YidC, these proteins may assist the folding of sizeable soluble domains of IMPs, thereby supporting their folding and oligomeric assembly. In conclusion, our analysis identifies many new IMPs/IMP complexes that depend on YidC for their biogenesis, responses that accompany depletion of YidC and an IMP characteristic that is associated with YidC dependence.     

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec-translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo-crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.     

Versatility of inner membrane protein biogenesis in Escherichia coli

To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far. The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains. We show that the assembly of the complete set of model IMPs is assisted (i.e. requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other. This indicates that IMP assembly is much more versatile than previously thought.     

Targeting, insertion, and localization of Escherichia coli YidC

YidC was recently shown to play an important role in the assembly of inner membrane proteins (IMPs) both in conjunction with and separate from the Sec-translocon. Little is known about the biogenesis and structural and functional properties of YidC, itself a polytopic IMP. Here we analyze the targeting and membrane integration of YidC using in vivo and in vitro approaches. The combined data indicate that YidC is targeted by the signal recognition particle and inserts at the SecAYEG-YidC translocon early during biogenesis, unlike its mitochondrial homologue Oxa1p. In addition, YidC is shown to be relatively abundant compared with other components involved in IMP assembly and is predominantly localized at the poles of the cell.     

Distinct requirements for translocation of the N-tail and C-tail of the Escherichia coli inner membrane protein CyoA

Inner membrane proteins (IMPs) of Escherichia coli use different pathways for membrane targeting and integration. YidC plays an essential but poorly defined role in the integration and folding of IMPs both in conjunction with the Sec translocon and as a Sec-independent insertase. Depletion of YidC only marginally affects the insertion of Sec-dependent IMPs, whereas it blocks the insertion of a subset of Sec-independent IMPs. Substrates of this latter "YidC-only" pathway include the relatively small IMPs M13 procoat, Pf3 coat protein, and subunit c of the F(1)F(0) ATPase. Recently, it has been shown that the steady state level of the larger and more complex CyoA subunit of the cytochrome o oxidase is also severely affected upon depletion of YidC. In the present study we have analyzed the biogenesis of the integral lipoprotein CyoA. Collectively, our data suggest that the first transmembrane segment of CyoA rather than the signal sequence recruits the signal recognition particle for membrane targeting. Membrane integration and assembly appear to occur in two distinct sequential steps. YidC is sufficient to catalyze insertion of the N-terminal domain consisting of the signal sequence, transmembrane segment 1, and the small periplasmic domain in between. Translocation of the large C-terminal periplasmic domain requires the Sec translocon and SecA, suggesting that for this particular IMP the Sec translocon might operate downstream of YidC.     

The ribosome and YidC. New insights into the biogenesis of Escherichia coli inner membrane proteins

In the bacterium Escherichia coli, inner membrane proteins (IMPs) are generally targeted through the signal recognition particle pathway to the Sec translocon, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. Lateral transport seems to be assisted by the IMP YidC. In this article, we discuss recent observations that point to a key role for the ribosome in IMP targeting and to the diverse roles of YidC in IMP assembly.     

Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs.     

Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli

The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Deltalon DeltaclpQ strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in E. coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.     

YidC and SecY mediate membrane insertion of a Type I transmembrane domain

YidC has been identified recently as an evolutionary conserved factor that is involved in the integration of inner membrane proteins (IMPs) in Escherichia coli. The discovery of YidC has inspired the reevaluation of membrane protein assembly pathways in E. coli. In this study, we have analyzed the role of YidC in membrane integration of a widely used model IMP, leader peptidase (Lep). Site-directed photocross-linking experiments demonstrate that both YidC and SecY contact nascent Lep very early during biogenesis, at only 50-amino acid nascent chain length. At this length the first transmembrane domain (TM), which acquires a type I topology, is not even fully exposed outside the ribosome. The pattern of interactions appears dependent on the position of the cross-linking probe in the nascent chain. Upon elongation, nascent Lep remains close to YidC and comes into contact with lipids as well. Our results suggest a role for YidC in both the reception and lipid partitioning of type I TMs.     

F(1)F(0) ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner membrane

Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration. We have examined the biogenesis of the F1F0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches. The data suggest that F0c is targeted by the SRP to the membrane, where it inserts at YidC in a Sec-independent mechanism. F0c appears to be the first natural substrate of this novel pathway.     

Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.     

YidC is involved in the biogenesis of anaerobic respiratory complexes in the inner membrane of Escherichia coli

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of this family have all been implicated in membrane protein biogenesis of aerobic respiratory and energy-transducing proteins. YidC is essential for the insertion of subunit c of the F(1)F(0)-ATP synthase and subunit a of cytochrome o oxidase. The aim of this study was to investigate whether YidC plays a role during anaerobic growth of Escherichia coli, specifically when either nitrate or fumarate are used as terminal electron acceptors or under fermentative conditions. The effect of YidC depletion on the growth, enzyme activities, and protein levels in the inner membrane was determined. YidC is essential for all anaerobic growth conditions tested, and this is not because of the decreased levels of F(1)F(0)-ATP synthase in the inner membrane only. The results suggest a role for YidC in the membrane biogenesis of integral membrane parts of the anaerobic respiratory chain.     

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.     

Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs-- or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.     

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.     

YidC is strictly required for membrane insertion of subunits a and c of the F(1)F(0)ATP synthase and SecE of the SecYEG translocase

YidC was previously discovered to play a critical role for the insertion of the Sec-independent M13 procoat and Pf3 coat phage proteins into the Escherichia coli inner membrane. To determine whether there is an absolute requirement of YidC for membrane protein insertion of any endogenous E. coli proteins, we investigated a few representative membrane proteins. We found that membrane subunits of the F(0) sector of the F(1)F(0)ATP synthase and the SecE protein of the SecYEG translocase are highly dependent on YidC for membrane insertion, based on protease mapping and immunoblot analysis. We found that the SecE dependency on YidC for membrane insertion does not contradict the observation that depletion of YidC does not block SecYEG-dependent protein export at 37 degrees C. YidC depletion does not decrease the SecE level low enough to block export at 37 degrees C. In contrast, we found that protein export of OmpA is severely blocked at 25 degrees C when YidC is depleted, which may be due to the decreased SecE level, as a 50% decrease in the SecE levels drastically affects protein export at the cold temperature [Schatz, P. J., Bieker, K. L., Ottemann, K. M., Silhavy, T. J., and Beckwith, J. (1991) EMBO J. 10, 1749-57]. These studies reported here establish that physiological substrates of YidC include subunits of the ATP synthase and the SecYEG translocase, demonstrating that YidC plays a vital role for insertion of endogenous membrane proteins in bacteria.