A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

Derman, Alan I.; Puziss, J W; Bassford, P J; Beckwith, Jon

doi:10.1002/j.1460-2075.1993.tb05728.x

Cited by 217 publications

(236 citation statements)

References 73 publications

Supporting

Mentioning

222

Contrasting

Unclassified

Order By: Relevance

“…It recently was found that MBPA2-26 and an analogous signal peptide-less PhoA species can be exported to the periplasm, albeit somewhat inefficiently, in cells harboring prlA4 (12). Thus, the ability of prlA666 and other prl alleles to promote the export of MBPA2-26 was investigated, using growth on maltose minimal medium as a qualitative assay for MBP export.…”

Section: Resultsmentioning

confidence: 99%

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations

1992

View full text Add to dashboard Cite

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prL4 (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prL4666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects.Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prUi mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide.In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prUA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.Proteins exported across the cytoplasmic membrane of Escherichia coli cells are usually synthesized with a cleavable, amino-terminal signal peptide, which is thought to be chiefly responsible for initiating the export process. This structure includes a central hydrophobic core followed by a somewhat less apolar signal peptidase processing site and preceded by a hydrophilic segment that exhibits a net positive charge due to the presence of one to three basic residues (39). The early mature region of such proteins generally lacks a net positive charge (40). For integral cytoplasmic membrane proteins, a region of net positive charge is usually found immediately adjacent to one end of each membrane-spanning stretch of hydrophobic amino acids (41). In both cases, the transmembrane orientation of the hydrophobic domains is thought to be primarily determined by the distribution of basic residues at either end, with the most positively charged end residing in the cytoplasm. This "positive-inside rule" was initially proposed by von Heijne (41).In support of the positive-inside rule, several studies have shown that systematically reducing the net charge of the signal peptide hydr...

show abstract

Section: Resultsmentioning

confidence: 99%

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations

1992

View full text Add to dashboard Cite

show abstract

“…Interestingly, an effect on the topology of model membrane proteins is shown for two prlA alleles (31,32). Secretory proteins completely lacking the signal, but not cytosolic proteins, are still exported by prlA mutants in a SecB-dependent manner (33). This is accounted for by the fact that SecB recognizes secretory proteins within their mature sequences (34,35) and thus targets them to the translocon.…”

Section: Sec61p Mutations Affect Protein Orientation In Distinct Ways-mentioning

confidence: 99%

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Junne

Schwede

Goder³

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.The Sec61/SecY translocon complex mediates translocation of proteins across and integration into the endoplasmic reticulum (ER) 3 membrane (1, 2). Proteins are targeted to this complex by hydrophobic signal sequences (3), and the signals and subsequent hydrophobic segments of the polypeptide are integrated into the bilayer as transmembrane helices. The translocon consists of Sec61␣ (Sec61p in yeast) with 10 transmembrane domains, and the single spanning proteins Sec61␤ (Sbh1p) and Sec61␥ (Sss1p), corresponding in bacteria to SecY/ SecG/SecE. The crystal structure of the SecY complex of the archaebacterium Methanococcus jannaschii (4) revealed a compact helix bundle that can form a hydrophilic channel with a lateral exit site. The central passage is blocked by a lumenal plug domain and a central constriction ring. To open, the plug needs to move out, and the ring must expand (5, 6). The crystal structure and cross-linking experiments (7) suggest that the translocation channel is formed by a single heterotrimer, although it has been proposed that upon opening of the channel two or more complexes may cooperate (8, 9).The simplest case of protein topogenesis is the orientation of a signal sequence upon entering the translocon. Cleavable signal sequences assume a N cyt /C exo orientation (cytoplasmic N and exoplasmic C terminus), whereas noncleavable signal-anchors of membrane proteins insert in N exo /C cyt or N cyt /C exo direction to translocate either their N-or their C-terminal end, respectively. Best established is the role of charged residues on either side of the hydrophobic core of the signal, which according to the "positive-inside rule" (10, 11) generally results in the more positive end to be cytosolic. Additional determinants are the folding of sequences N-terminal to an internal signal that may sterically hinder N-translocation (12) and the ...

show abstract

“…Therefore, in the case of the intermembrane shuttle, an import sequence-independent internalization of StAR into the mitochondrial intermembrane space is necessary and could be possible (Derman et al 1993). In the event where StAR functions outside mitochondria, StAR interactions with mitochondrial proteins are key for proper StAR function and still remain to be clearly identified.…”

Section: The Pros and Consmentioning

confidence: 99%

Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR

Mathieu¹,

Fleury²,

Ducharme³

et al. 2002

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783·9 Å 2 in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal α-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.

show abstract

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

Cited by 217 publications

References 73 publications

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

Insights into steroidogenic acute regulatory protein (StAR)-dependent cholesterol transfer in mitochondria: evidence from molecular modeling and structure-based thermodynamics supporting the existence of partially unfolded states of StAR

Contact Info

Product

Resources

About