Suppressor mutations that restore export of a protein with a defective signal sequence

doi:10.1016/0092-8674(81)90272-5

Cell

Volume 23, Issue 1, January 1981, Pages 79-88

https://doi.org/10.1016/0092-8674(81)90272-5 Get rights and content

Abstract

A selection procedure is described that should allow the genetic identification of cellular components involved in the process of protein localization in Escherichia coli. This procedure makes use of mutations that alter the signal sequence of the λ receptor protein (product of the lamB gene), and prevent export of this protein to its normal outer membrane location. Several suppressor mutations have been identified that restore export of the mutant λ receptor protein. Mapping experiments show that the suppressor phenotype is the result of mutations in any of at least three different chromosomal loci. One class of suppressor mutations, the class containing the largest number of independent isolates, maps in the major ribosomal gene cluster, suggesting that the suppressor phenotype is the consequence of an altered ribosomal protein. This class of suppressors phenotypically suppresses all known export-defective mutations, internal to the signal sequence region of the lamB gene. These results suggest that ribosomes play an important role in the export of λ receptor to the outer membrane.

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The Bacteriophage Lambda

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Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling
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The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.
Cracking outer membrane biogenesis
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Citation Excerpt :
Since these mutant proteins are retained within the cytoplasm, E. coli expressing these mutant proteins cannot grow using maltose or maltodextrins, respectively, as the sole source of carbon. Dominant, gain-of-function mutations that secrete the mutant proteins despite their defective signal sequences were selected for by demanding growth on maltose or maltodextrin minimal medium [136–138]. This and the above selection isolated mutations that mapped to the same genetic loci [139,140].
The outer membrane is a distinguishing feature of the Gram-negative envelope. It lies on the external face of the peptidoglycan sacculus and forms a robust permeability barrier that protects extracytoplasmic structures from environmental insults. Overcoming the barrier imposed by the outer membrane presents a significant hurdle towards developing novel antibiotics that are effective against Gram-negative bacteria. As the outer membrane is an essential component of the cell, proteins involved in its biogenesis are themselves promising antibiotic targets. Here, we summarize key findings that have built our understanding of the outer membrane. Foundational studies describing the discovery and composition of the outer membrane as well as the pathways involved in its construction are discussed.
Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum
2020, Molecular and Biochemical Parasitology
Citation Excerpt :
Although multiple selection markers are available for use in P. falciparum [31], we show that the split-DHFR approach can be used to simultaneously select for two plasmids using a single drug selection marker, thus leaving a large range of selection markers for subsequent manipulations. Additionally, genetic screens in model organisms have in many cases utilised selection markers that are only active in a particular cellular compartment [32–34], thus allowing identification of genes required for protein localisation or trafficking. As reconstitution of split-DHFR requires that both fragments be localised in the same cellular compartment, it may be adapted for use in similar screening strategies in the malaria parasite.
Defining protein-protein interactions is fundamental to the understanding of gene function. Protein-fragment complementation assays have been used for the analysis of protein-protein interactions in various organisms. The split-dihydrofolate reductase (DHFR) protein-fragment complementation assay utilises two complementary fragments of the enzyme fused to a pair of potentially interacting proteins. If these proteins interact, the DHFR fragments associate, fold into their native structure, reconstitute their function and confer resistance to antifolate drugs. We show that murine DHFR fragments fused to interacting proteins reconstitute a functional enzyme and confer resistance to the antifolate drug WR99210 in Plasmodium falciparum. These data demonstrate that the split-DHFR method can be used to detect in vivo protein-protein interactions in the parasite. Additionally, we show that split-DHFR fragments can be used as selection markers, permitting simultaneous selection of two plasmids in the presence of a single antifolate drug. Taken together, these experiments show that split-DHFR represents a valuable tool for the characterisation of Plasmodium protein function and genetic manipulation of the parasite.
Interaction mapping of the Sec61 translocon identifies two Sec61 regions interacting with hydrophobic segments in translocating chains
2018, Journal of Biological Chemistry
Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them.
Unlocking the Bacterial SecY Translocon
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Citation Excerpt :
This in turn displaces the plug (Figure 1A, right panels, red helix), and likely primes the channel for translocation. Mutations of SecY have been identified that allow in vivo transport of pre-proteins with a defective SS (prlA mutants) (Smith et al., 2005; Emr et al., 1981; Derman et al., 1993). These mutants also exhibit increased translocation activity and are not further stimulated by the PMF (Nouwen et al., 1996).
The Sec translocon performs protein secretion and membrane protein insertion at the plasma membrane of bacteria and archaea (SecYEG/β), and the endoplasmic reticular membrane of eukaryotes (Sec61). Despite numerous structures of the complex, the mechanism underlying translocation of pre-proteins, driven by the ATPase SecA in bacteria, remains unresolved. Here we present a series of biochemical and computational analyses exploring the consequences of signal sequence binding to SecYEG. The data demonstrate that a signal sequence-induced movement of transmembrane helix 7 unlocks the translocon and that this conformational change is communicated to the cytoplasmic faces of SecY and SecE, involved in SecA binding. Our findings progress the current understanding of the dynamic action of the translocon during the translocation initiation process. The results suggest that the converging effects of the signal sequence and SecA at the cytoplasmic face of SecYEG are decisive for the intercalation and translocation of pre-protein through the SecY channel.
Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon
2024, EMBO Journal

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Abstract

References (35)

Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage λ receptor

J. Mol. Biol.

Identification and organization of ribosomal protein genes of Escherichia coli carried by λfus2 transducing phage

J. Biol. Chem.

The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity

J. Mol. Biol.

Ribosomes from Escherichia coli merodiploids heterozygous for resistance to streptomycin and to spectinomycin

J. Mol. Biol.

Linkage map of Escherichia coli K-12

Intracellular protein topogenesis

Transfer of proteins across membranes. I. Presence of proteolytically processed and nonprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

J. Cell Biol.

Translocation of proteins across membranes: the signal hypothesis and beyond

The mechanism of protein secretion across membranes

Nature

Mutations altering the cellular localization of the phage λ receptor, an Escherichia coli outer membrane protein

A mechanism of protein localization: the signal hypothesis and bacteria

J. Cell Biol.

Sequence analysis of mutations that prevent export of λ receptor, an Escherichia coli outer membrane protein

Nature

The contrasting role of strA and ram gene products in ribosomal functioning

Physical mapping of TS1 by restriction endonucleases

Genetic analysis of the maltose A region in Escherichia coli

J. Bacteriol.

DNA sequence encoding the NH₂-terminal peptide involved in transport of λ receptor, an E. coli secretory protein

The Bacteriophage Lambda

Cited by (246)

Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling

Cracking outer membrane biogenesis

Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum

Interaction mapping of the Sec61 translocon identifies two Sec61 regions interacting with hydrophobic segments in translocating chains

Unlocking the Bacterial SecY Translocon

Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon

Cell

ArticleSuppressor mutations that restore export of a protein with a defective signal sequence

Abstract

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Linkage map of Escherichia coli K-12

Intracellular protein topogenesis

Transfer of proteins across membranes. I. Presence of proteolytically processed and nonprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

J. Cell Biol.

Translocation of proteins across membranes: the signal hypothesis and beyond

The mechanism of protein secretion across membranes

Nature

Mutations altering the cellular localization of the phage λ receptor, an Escherichia coli outer membrane protein

A mechanism of protein localization: the signal hypothesis and bacteria

J. Cell Biol.

Sequence analysis of mutations that prevent export of λ receptor, an Escherichia coli outer membrane protein

Nature

The contrasting role of strA and ram gene products in ribosomal functioning

Physical mapping of TS1 by restriction endonucleases

Genetic analysis of the maltose A region in Escherichia coli

J. Bacteriol.

DNA sequence encoding the NH2-terminal peptide involved in transport of λ receptor, an E. coli secretory protein

The Bacteriophage Lambda

Article
Suppressor mutations that restore export of a protein with a defective signal sequence

DNA sequence encoding the NH₂-terminal peptide involved in transport of λ receptor, an E. coli secretory protein