Efficient Protein Translocation Research Articles

Cytosolic delivery of monobodies using the bacterial type III secretion system inhibits oncogenic BCR: ABL1 signaling

BackgroundThe inability of biologics to pass the plasma membrane prevents their development as therapeutics for intracellular targets. To address the lack of methods for cytosolic protein delivery, we used the type III secretion system (T3SS) of Y. enterocolitica, which naturally injects bacterial proteins into eukaryotic host cells, to deliver monobody proteins into cancer cells. Monobodies are small synthetic binding proteins that can inhibit oncogene signaling in cancer cells with high selectivity upon intracellular expression. Here, we engineered monobodies targeting the BCR::ABL1 tyrosine kinase for efficient delivery by the T3SS, quantified cytosolic delivery and target engagement in cancer cells and monitored inhibition of BCR::ABL1 signaling.MethodsIn vitro assays were performed to characterize destabilized monobodies (thermal shift assay and isothermal titration calorimetry) and to assess their secretion by the T3SS. Immunoblot assays were used to study the translocation of monobodies into different cell lines and to determine the intracellular concentration after translocation. Split-Nanoluc assays were performed to understand translocation and degradation kinetics and to evaluate target engagement after translocation. Phospho flow cytometry and apoptosis assays were performed to assess the functional effects of monobody translocation into BCR:ABL1-expressing leukemia cells.ResultsTo enable efficient translocation of the stable monobody proteins by the T3SS, we engineered destabilized mutant monobodies that retained high affinity target binding and were efficiently injected into different cell lines. After injection, the cytosolic monobody concentrations reached mid-micromolar concentrations considerably exceeding their binding affinity. We found that injected monobodies targeting the BCR::ABL1 tyrosine kinase selectively engaged their target in the cytosol. The translocation resulted in inhibition of oncogenic signaling and specifically induced apoptosis in BCR::ABL1-dependent cells, consistent with the phenotype when the same monobody was intracellularly expressed.ConclusionHence, we establish the T3SS of Y. enterocolitica as a highly efficient protein translocation method for monobody delivery, enabling the selective targeting of different oncogenic signaling pathways and providing a foundation for future therapeutic application against intracellular targets.

DARPins bind their cytosolic targets after having been translocated through the protective antigen pore of anthrax toxin

Intracellular protein–protein interactions in aberrant signaling pathways have emerged as a prime target in several diseases, particularly cancer. Since many protein–protein interactions are mediated by rather flat surfaces, they can typically not be interrupted by small molecules as they require cavities for binding. Therefore, protein drugs might be developed to compete with undesired interactions. However, proteins in general are not able to translocate from the extracellular side to the cytosolic target site by themselves, and thus an efficient protein translocation system, ideally combining efficient translocation with receptor specificity, is in high demand. Anthrax toxin, the tripartite holotoxin of Bacillus anthracis, is one of the best studied bacterial protein toxins and has proven to be a suitable candidate for cell-specific translocation of cargoes in vitro and in vivo. Our group recently developed a retargeted protective antigen (PA) variant fused to different Designed Ankyrin Repeat Proteins (DARPins) to achieve receptor specificity, and we incorporated a receptor domain to stabilize the prepore and prevent cell lysis. This strategy had been shown to deliver high amounts of cargo DARPins fused behind the N-terminal 254 amino acids of Lethal Factor (LFN). Here, we established a cytosolic binding assay, demonstrating the ability of DARPins to refold in the cytosol and bind their target after been translocated by PA.

Signal sequences encode information for protein folding in the endoplasmic reticulum.

One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon. How protein translocation coordinates with chaperone availability in the ER to promote protein folding remains unclear. We find that marginally hydrophobic signal sequences and transmembrane domains cause transient retention at the Sec61 translocon and require the luminal BiP chaperone for efficient protein translocation. Using a substrate-trapping proteomic approach, we identify that nascent proteins bearing marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon. Sec63 is co-translationally recruited to the translocation site and mediates BiP binding to incoming polypeptides. BiP binding not only releases translocationally paused nascent chains but also ensures protein folding in the ER. Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent translocation, but translocated proteins are prone to misfold and aggregate in the ER under limited BiP availability. Thus, the signal sequence-guided protein folding may explain why signal sequences are diverse and use multiple protein translocation pathways.

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita. The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof.

Control of Enzyme Reaction Initiation inside Giant Unilamellar Vesicles by the Cell-Penetrating Peptide-Mediated Translocation of Cargo Proteins.

Cell-penetrating peptides (CPPs) play important roles in directly delivering biomolecules, such as DNA, proteins, and peptides, into living cells. In artificial lipid membranes, such as planar lipid bilayers, the direct membrane translocation of β-galactosidase via Pep-1 (one of the CPPs) is dependent upon a voltage gradient between the inner and outer leaflets of the lipid membranes. Giant unilamellar vesicles (GUVs) with asymmetric lipid distributions, which are recently generated using microfluidic technologies, can be observed by optical microscopy. Therefore, interactions between CPPs and asymmetric lipid bilayers in different kinds of lipids and the translocation mechanism of proteins via CPPs into GUVs can be investigated at the level of a single asymmetric GUV. This CPP-based system for transporting proteins into GUVs will be applied to control the start of enzyme reactions in GUVs. This study aimed to explore efficient protein translocation into GUVs via CPP and demonstrate that enzymatic reactions start in GUVs using a CPP-mediated direct translocation. The interactions and the enzyme reactions between the CPP (Pep-1 or penetratin)-DNase I complexes and the asymmetric or symmetric GUV membranes containing the negatively or neutrally charged lipids were observed by confocal laser-scanning microscopy. The asymmetric GUVs containing phosphatidylserine (PS) in the inner leaflet showed efficient DNase I translocation into GUVs via penetratin. Finally, the formation of a cross-linked actin network was observed in asymmetric PS GUVs incubated with Pep-1-streptavidin complexes. The CPP-mediated direct translocation can contribute to developing artificial cell models with the capacity to control the initiation of enzymatic reactions.

Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

Inactivation of HIV-1 in Polarized Infant Tonsil Epithelial Cells by Human Beta-Defensins 2 and 3 Tagged with the Protein Transduction Domain of HIV-1 Tat.

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT.

InVivo Dissection of the Intrinsically Disordered Receptor Domain of Tim23

In the intermembrane space (IMS) of mitochondria, the receptor domain of Tim23 has an essential role during translocation of hundreds of different proteins from the cytosol via the TOM and TIM23 complexes in the outer and inner membranes, respectively. This intrinsically disordered domain, which can even extend into the cytosol, was shown, mostly in vitro, to interact with several subunits of the TOM and TIM23 complexes. To obtain molecular understanding of this organizational hub in the IMS, we dissected the IMS domain of Tim23 in vivo. We show that the interaction surface of Tim23 with Tim50 is larger than previously thought and reveal an unexpected interaction of Tim23 with Pam17 in the IMS, impairment of which influences their interaction in the matrix. Furthermore, mutations of two conserved negatively charged residues of Tim23, close to the inner membrane, prevented dimerization of Tim23. The same mutations increased exposure of Tim23 on the mitochondrial surface, whereas dissipation of membrane potential decreased it. Our results reveal an intricate network of Tim23 interactions in the IMS, whose influence is transduced across two mitochondrial membranes, ensuring efficient translocation of proteins into mitochondria.

Increased expression of the bacterial glycolipid MPIase is required for efficient protein translocation across membranes in cold conditions

Protein integration into and translocation across biological membranes are vital events for organismal survival and are fundamentally conserved among many organisms. Membrane protein integrase (MPIase) is a glycolipid that drives membrane protein integration into the cytoplasmic membrane in Escherichia coli MPIase also stimulates protein translocation across the membrane, but how its expression is regulated is incompletely understood. In this study, we found that the expression level of MPIase significantly increases in the cold (<25 °C), whereas that of the SecYEG translocon does not. Using previously created gene-knockout E. coli strains, we also found that either the cdsA or ynbB gene, both encoding rate-limiting enzymes for MPIase biosynthesis, is responsible for the increase in the MPIase expression. Furthermore, using pulse-chase experiments and protein integration assays, we demonstrated that the increase in MPIase levels is important for efficient protein translocation, but not for protein integration. We conclude that MPIase expression is required to stimulate protein translocation in cold conditions and is controlled by cdsA and ynbB gene expression.

A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import.

Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins.

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these “molecular syringes” for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells.

Cooperation of TOM and TIM23 Complexes during Translocation of Proteins into Mitochondria

Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM–TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM–TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein–protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes.

Pam17 and Tim44 act sequentially in protein import into the mitochondrial matrix

Import of proteins into the matrix is driven by the Tim23 presequence translocase-associated import motor PAM. The core component of PAM is the mitochondrial chaperone mtHsp70, which ensures efficient translocation of proteins across the inner membrane through interactions with the J-protein complex Pam16–Pam18 (Tim16–Tim14) and its cochaperone Tim44. The recently identified non-essential Pam17 is a further member of PAM. Genetic and biochemical analyses reveal synthetic interactions between PAM17 and TIM44. Pam17 is involved in an early stage of protein translocation whereas Tim44 assists in a later step of transport, suggesting that both proteins can cooperate in a complementary manner in protein import.

Residues of Tim44 Involved in both Association with the Translocon of the Inner Mitochondrial Membrane and Regulation of Mitochondrial Hsp70 Tethering

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.

SRP Keeps Polypeptides Translocation-Competent by Slowing Translation to Match Limiting ER-Targeting Sites

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.

The Plug Domain of Yeast Sec61p Is Important for Efficient Protein Translocation, but Is Not Essential for Cell Viability

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus

The heterotetrameric Sec62/63 complex associates with the heterotrimeric Sec61 complex to form the heptameric Sec complex. This complex is necessary and sufficient for post-translational protein translocation across the membrane of the endoplasmic reticulum. We show that Sec63p is phosphorylated at its C-terminal domain by the protein kinase CK2 and that this phosphorylation strengthens the interaction between the cytosolic domains of Sec63p and Sec62p. Exchanging either threonine 652 or threonine 654 against the nonphosphorylatable alanines in Sec63p impairs the binding to Sec62p and interferes with the efficient translocation of proteins across the membrane of the endoplasmic reticulum. These findings show that phosphorylation of Sec63p is required for tightly recruiting the putative signal-sequence-binding subunit Sec62p to the Sec complex.

Nucleotide exchange from the high-affinity ATP-binding site in SecA is the rate-limiting step in the ATPase cycle of the soluble enzyme and occurs through a specialized conformational state.

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.

Regulation of lipid composition in biological membranes—biophysical studies of lipids and lipid synthesizing enzymes

The study of the role played by membrane lipids, functional lipidomics, has become increasingly important in membrane biology. The physico-chemical properties of the lipids in biological membranes are subject to some fundamental requirements. In general, the acyl chains shall be in a liquid-like state to keep the membrane proteins active, and the lipids must form a bilayer structure in order for the membrane to be an insulating barrier. However, a potential ability of the lipids to form nonbilayer structures seems to be a prequisite for several membrane-associated cell processes. Therefore, organisms exposed to changes in the environmental conditions, such as temperature and uptake of fatty acids, adjust their membrane lipid composition. Examples of prokaryotic organisms that have been studied in this respect are the cell wall-less bacterium Acholeplasma laidlawii, the Gram-negative bacterium Escherichia coli, and the Gram-positive bacteria Bacillus megaterium and Clostridium butyricum, and among eukaroytic organisms are found fungi, higher plants, and poikilothermic animals. By synthesizing a proper combination of acyl chain and polar head group structures, the organisms modify the phase transition temperatures of the membrane lipids so that they are maintained in a lamellar liquid crystalline phase, and the formation of a lamellar gel phase as well as reserved nonlamellar phases is avoided. It has been shown that A. laidlawii and E. coli maintain a balance between lamellar-formed and nonlamellar-forming lipids. A growing body of evidence shows that nonlamellar-forming membrane lipids play essential roles in many aspects of membrance functioning. Short-lived nonbilayer structures are probably formed in the processes of fusion and fission of lipid bilayers, and long-lived bilayer structures with a small radius of curvature occur in several types of biological membranes (e.g. smooth endoplasmic reticulum, inner mitochondrial membrane, and prolamellar bodies). The activity of membrane-associated proteins can be modulated by adding detergents or nonlamellar-forming lipids to bilayers. Some examples are the regeneration of denatured bacteriorphodopsin, and the activities of protein kinase C, some phospholipases, and some key lipid synthases involved in the lipid metabolism of eukaryotic cells, A. laidlawii, and E. coli. The physico-chemical properties of the lipid matrix can be a direct feed-back signal on the activity of the lipid synthases. Finally, nonlamellar-forming lipids are essential for a proper cell division, and an efficient translocation of proteins across the plasma membrane, of E. coli cells.