Function of Positive Charges Following Signal-Anchor Sequences during Translocation of the N-terminal Domain

Kida, Yuichiro; Morimoto, Fumiko; Mihara, Katsuyoshi; Sakaguchi, Masao

doi:10.1074/jbc.m506613200

Cited by 24 publications

(28 citation statements)

References 28 publications

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“…Overall, our results show (i) that flanking Lys and Arg residues contribute equally to the apparent free energy of membrane insertion (⌬G app ), (ii) that the effect is maximal when the charged residues are placed a few residues away from the H-segment, (iii) that a hydrophobic residue added immediately downstream of a contiguous stretch of n Lys residues contributes favorably to ⌬G app only if n Յ 3, (iv) that a helix-breaking Pro residue reduces the contribution to ⌬G app from downstream positively charged residues, and (v) that a stretch of 3 or 6 consecutive Lys residues can contribute significantly to ⌬G app from a distance of up to Ϸ13 residues away from the hydrophobic segment. There is a striking similarity between these result and the effect exerted by positively charged residues on the orientation of transmembrane ␣-helices, where stretches of Lys or Arg residues have been shown to promote a cytosolic location when located up to Ϸ25 residues away from the hydrophobic transmembrane segment (23,24).…”

Section: Discussionmentioning

confidence: 69%

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Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum

Lerch-Bader

Lundin²,

Kim³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Positively charged residues located near the cytoplasmic end of hydrophobic segments in membrane proteins promote membrane insertion and formation of transmembrane ␣-helices. A quantitative understanding of this effect has been lacking, however. Here, using an in vitro transcription-translation system to study the insertion of model hydrophobic segments into dog pancreatic rough microsomes, we show that a single Lys or Arg residue typically contributes approximately ؊0.5 kcal/mol to the apparent free energy of membrane insertion (⌬G app) when placed near the cytoplasmic end of a hydrophobic segment and that stretches of 3-6 Lys residues can contribute significantly to ⌬Gapp from a distance of up to Ϸ13 residues away. membrane protein ͉ positive inside rule ͉ hydrophobicity scale ͉ translocon T he biosynthesis of ␣-helical membrane proteins requires their insertion, folding, and oligomerization in the target membrane. In eukaryotic cells, most membrane proteins insert cotranslationally into the endoplasmic reticulum (ER) membrane in a process mediated by the heterotrimeric Sec61 translocon (1, 2).In a series of recent studies (3-6), we have determined the sequence characteristics responsible for the insertion of a transmembrane helix into the ER membrane by measuring the membrane-insertion efficiency of designed hydrophobic segments (H-segments) engineered into a model protein. The position-dependent apparent free energy of insertion derived from these studies for the different amino acids provides the basis for a truly ''biological'' hydrophobicity scale (6). The biological scale correlates well with biophysical and statistical hydrophobicity scales, such as the Wimley-White water-octanol partitioning scale (7) and Sansom's structure-based statistical scale (8). This correlation suggests that the insertion of transmembrane helices is largely determined by the thermodynamics of protein-lipid interactions (3), which is not unreasonable given the structure of the archeal Sec61 translocon in which a dynamic ''lateral gate'' may provide a polypeptide in transit ready access to the lipid bilayer (9, 10).The biosynthesis of membrane proteins, however, not only requires the proper insertion of the transmembrane segments but also their correct orientation in the membrane, and the translocon is thought to play a pivotal role in this process as well (reviewed in ref. 11). Positively charged residues in membrane proteins are found predominantly on the cytoplasmic side of the membrane [the ''positive inside'' rule (12, 13)], flanking either signal sequences (14) or transmembrane segments (12). Consistent with this rule, positively charged residues favor insertion when placed at the cytoplasmic end of a transmembrane segment (6).To characterize more fully the effects on membrane insertion of charged flanking residues, we have now measured the apparent free energy of membrane insertion of a set of H-segments with different combinations of charged flanking residues engineered into a model membrane protein. We have tested the ef...

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Section: Discussionmentioning

confidence: 69%

“…In particular, charged flanking residues have been shown to either reduce or enhance membrane insertion, with luminal negatively charged or strongly polar flanking residues reducing insertion and cytosolic positively charged residues enhancing insertion (6,23).…”

Section: Discussionmentioning

confidence: 99%

Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum

Lerch-Bader

Lundin²,

Kim³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, in the case of synaptotagmin II, there are eight positive charges just after the H-segment. When they are moved more than 20 residues downstream, they still affect the orientation of the H-segment, whereas those that are moved more than 30 residues downstream no longer affect the orientation (Kida et al, 2006). In many cases, the TM topology of a signal sequence is likely to be determined within a short range.…”

Section: Discussionmentioning

confidence: 99%

“…The H-segment of the signal sequence penetrates the translocon, and then one side of the H-segment moves into the lumen to form the TM topology. During the insertion into the translocon, the orientation of the signal sequence is determined by the flanking positively charged amino acid residues (Goder and Spiess, 2001;Kida et al, 2006;Sakaguchi, 1997). If the Nterminal side of the H-segment is rich in positive charges, as observed in signal peptide and type II signal-anchor sequences, the N-terminus is retained on the cytoplasmic side and the segment forms the N cytosol and C lumen orientation (termed the type II orientation).…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, if the Cterminal side is rich in positive charges, the N-terminus is translocated through the translocon and forms the N lumen and C cytosol orientation (the type I orientation) and the signal-anchor is retained as a TM segment. For example, the topology of synaptotagmin II is gradually converted depending on the number of positive charges downstream of the H-segment (Kida et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Positive charges on the translocating polypeptide chain arrest movement through the translocon

Fujita

Yamagishi

Kida

et al. 2011

Journal of Cell Science

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SummaryPolypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed.

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Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

et al. 2007

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Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476-G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.

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Function of Positive Charges Following Signal-Anchor Sequences during Translocation of the N-terminal Domain

Cited by 24 publications

References 28 publications

Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum

Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum

Positive charges on the translocating polypeptide chain arrest movement through the translocon

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

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