Institute for Biochemistry II, Medical Faculty of the Friedrich Schiller University Jena, Nonnenplan 2, D-07743 Jena, Germany
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Abstract |
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Keywords: amino acid composition/C-terminal bias/C-terminus/phospholipid binding/SIP18
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Introduction |
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It is well known from statistical analysis that the occurrence of certain amino acids at a protein C-terminus is non-random as was shown for Escherichia coli, S.cerevisiae and human proteins. The composition of the C-terminal peptide sequence differs from that expected for the overall amino acid composition of a given protein. In general, it was shown that amino acids which are positively charged at physiological pH values are over-represented in the C-terminal peptide sequence while Gly residues are under-represented (Berezovsky et al., 1997). Explanations for this C-terminal bias are taken from the fact that C-termini of proteins possess some distinct properties.
First, the C-terminal amino acid residues could interfere with translation termination signals at the ribosome machinery itself. This has been shown for E.coli where the composition of the C-terminal end of the nascent peptide influences translation termination with the strongest contributing effects from the last amino acid (1 location) and the penultimate amino acid (2 location) (Bjornsson et al., 1996). In yeast and human cells, too, the nucleotide stop codon context, including regions upstream of the stop codon which encode C-terminal amino acids, influences the efficacy of stop codon recognition and translation termination (Bonetti et al., 1995
; McCaughan et al., 1995
). However, effects in yeast seem to be of lesser extent compared to effects in E.coli or Bacillus subtilis (Mottagui-Tabar et al., 1998
; for overview see Bertram et al., 2001
).
Secondly, it was proposed, that charged amino acids concentrated in the C-terminal region of a protein will stabilize its overall spatial structure by fixation of the terminal tail to the core of the protein due to electrostatic interactions. Even if these effects are not observed in matured proteins the charged termini could be important during the folding process of the nascent polypeptide chain (Christopher and Baldwin, 1996). For collagen it was shown that the carboxy end stabilizes the spatial protein structure (Prockop and Kivirikko, 1995
) but most crystallization experiments fail to resolve exactly the location of the N- and C-terminal parts of proteins leaving this question unanswered.
Thirdly, an alternative explanation of the C-terminal bias of positively charged amino acids could be a possible interaction of charged amino acid residues not only with inner protein structures themselves but with components of the cellular environment such as nucleic acids or membranes. The interaction of proteins with membranes can occur in two different manners. On the one hand, hydrophobic amino acid residues could contribute to membrane attachment by insertion into the inner core of the lipid bilayer. The other possibility would be provided by positively charged amino acids which are found predominantly in C-terminal peptides and which can directly interact with negatively charged components of the membrane. These electrostatic interactions are thought to be less stable than hydrophobic interactions and could be the first step of the final attachment provided by hydrophobic residues. Particular emphasis is placed on the interactions of proteins with phospholipids acting as second messengers in signalling pathways, mainly phosphoinositides or inositol polyphosphates. The first phospholipid-binding domain characterized was the pleckstrin homology (PH) domain (Harlan et al., 1994). PH domains have been found in proteins from numerous species from mammals to yeast, but neither in plants nor in bacteria. Comparing all putative PH domains found so far it is obvious that they show very low sequence similarity. Other lipid-binding domains are C2 domains, SH2 domains (Bottomley et al., 1998
) and the recently described FYVE domain (Gaullier et al., 1998
). What is the molecular basis for proteinphosphoinositide interaction? Many of the binding sites were mapped to assign the phosphoinositide binding to separate regions in the protein. These collinear sequences are mostly rich in basic and hydrophobic amino acid residues or characterized by lysine/arginine-rich sequences (for overview see Martin, 1998
; Hurley and Misra, 2000
). In general, the isolated lipid-binding motifs with lengths from 10 to approximately 120 amino acid residues in binding studies functioned similar to the entire protein wherein lysine and arginine residues accounted for lipid binding as proven by mutational analysis (Janmey et al., 1992
; Jorgensen et al., 1995
). Conserved lysine and arginine residues are found in the FYVE domain (Fruman et al., 1999
) as well as in the SH and PH domains (Isakoff et al., 1998
; Kang et al., 2000
).
The above considerations prompted us to examine whether one additional explanation for the C-terminal bias is the anchorage of C-termini to membrane components, especially to positively charged phospholipids.
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Materials and methods |
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DNA restriction and modifying enzymes were from Boehringer Mannheim (Mannheim, Germany) and New England BioLabs (Frankfurt, Germany). GlutathioneSepharose 4B was purchased from Amersham Pharmacia Biotech AB (Freiburg, Germany), the DNA sequencing kit-LC from Epicentre Technologies (Madison, WI, USA). Antibodies were from Quantum Biotechnologies (anti-AFP mAB, 11E5) (Heidelberg, Germany) and Sigma (anti-glutathione-S-transferase IgG) (München, Germany). Phospholipids were from Sigma and rhodamine-phosphatidylethanolamine (Rh-PE) from Avanti Polar Lipids (Alabaster, AL, USA).
Database for yeast proteins
All known S.cerevisiae protein sequences were extracted from the YPDTM database (http://www.proteome.com/databases/). In total, C-terminal sequences of 6235 yeast proteins were analysed for the overall occurrence of amino acid residues at the last (1) sense codon position as well as the penultimate (2) position and position (3) without further statistical considerations. The occurrence of any amino acid residue or combinations of residues are given in absolute numbers or in percentage of all yeast proteins (protein positions) from the database.
Library construction and purification of GST-fusion proteins
For library construction, S.cerevisiae genomic DNA was partially digested with Sau3AI and 110 kb fragments were isolated and ligated to BamHI-cut pEG(KT) (Mitchell et al., 1993). The ligation mixture was transformed into E.coli and approximately 15 000 independent colonies were obtained. DNA was prepared from a batch of all 15 000 E.coli colonies. Forty single clones were selected to characterize the expression library for insert size and number of insert-containing plasmids. Ninety-six percent of plasmids were found to contain inserts of ~15 kb. The haploid S.cerevisiae strain BJ5459 (Mata ura3-52 trp1 lys2-801 leu2
1 his3
200 pep4::HIS3 prb1
1.6R can1) was transformed with this library and approximately 8000 independent colonies were obtained. 4000 single yeast colonies were replated onto master plates, finally 3800 of them were tested for lipid binding of the expressed GST-fusion protein (see Preparation of lipid vesicles and lipid-binding assay). For preparation of GST-fusion proteins yeast colonies were grown at 30°C in galactose-containing medium to induce expression of GST-fusion proteins. After 20 h, cells were harvested by centrifugation, resuspended in ice-cold lysis buffer (10 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 1 mM PMSF, 10 mM benzamidine), transferred to 1.5 ml test tubes, and 1 volume of glass beads (
ISOdia/ 0.5 mm) was added. Cell lysis was performed by vigorous shaking for 20 min at 4°C. Lysates were centrifuged for 5 min at 20 000 g and supernatants were incubated with glutathioneSepharose beads. After incubation for 1 h at 4°C Sepharose beads were washed several times to obtain the immobilized GST-fusion protein. The molecular mass, purity and concentration of the proteins were estimated by SDSPAGE gel.
Preparation of lipid vesicles and lipid-binding assay
Mixed liposomes were prepared as previously described (Soom et al., 2001). In brief, phospholipids (from bovine brain) and Rh-PE were dissolved in chloroform:methanol (1:1). Phospholipid mixtures contained 15% phosphatidylserine, 40% phosphatidylinositol, 40% phosphatidylinositolphosphates and 5% phosphatidylinositolbisphosphates. The content of phospholipids was verified by HPTLC separation as previously described (Mayer et al., 2000
). Phospholipid mixtures were dried under N2 and resolved in 10 mM HEPES buffer (pH 7.4; 100 mM NaCl). After homogenizing and freezing/thawing, mixtures were sonicated and centrifuged. Supernatants containing small unilamellar vesicles were used in binding assays with glutathioneSepharose immobilized proteins.
Lipid-binding assays were performed as described previously (Soom et al., 2001). For the binding assay, immobilized GST-fusion proteins (~1 µg/sample) and small unilamellar vesicles (500 µg phospholipid/ml; 50 µl/sample) were mixed in binding buffer to a total volume of 100 µl and incubated with agitation at 22°C for 30 min. The Sepharose was spun down and washed with binding buffer. Liposome binding to immobilized proteins was quantified by fluorescence measurement in 96-well plates (Fluoroscan II; Laborsystems GmbH, Frankfurt, Germany) using 390 and 590 nm, respectively, as excitation and emission wavelengths. Data presented were corrected for non-specific binding by subtracting the fluorescence found for glutathioneSepharose alone. GSTphosphatidylinositol 3-kinase
(PI3K
), which binds anionic phospholipids including phosphoinositides (Kirsch et al., 2001
), was taken as control.
SIP18 constructs
The S.cerevisiae SIP18 gene was amplified by PCR from genomic DNA. Point mutations for subcloning and truncation of expressed protein were generated by overlap extension PCR and all constructs were verified by sequencing. GST-fusion constructs were inserted into the plasmid pEG(KT) (see Library construction and purification of GST-fusion proteins) and the yeast strain BJ5459 was transformed with the plasmids.
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Results |
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We examined all S.cerevisiae proteins predicted by open reading frames (ORFs) in the yeast genome database. Analysis reveals an absolute preference for lysine in both the (1) and (2) location of the C-terminal peptide. Also, at position (3) Lys was still over-represented (Figure 1). Twelve percent of all yeast proteins show Lys at the C-terminal position (1) whereas the frequency at other positions decreases with increasing distance from the C-terminus: 11% Lys at position (2); 9% Lys at position (3); 10% Lys at position (4). Phospholipid-binding peptides often contain stretches of basic amino acid residues (Fruman et al., 1999
; Xue et al., 1999
; Hurley and Misra, 2000
). Apparently, positively charged amino acid residues may contribute to phospholipid binding by electrostatic interactions. For this reason we investigated the frequency of appearance of all possible combinations of amino acids at the last two positions (Table I
). Overall analysis reveals a high predominance of the paired residues Lys (2)Lys(1)stop which was found 151 times, followed by Ser(2)Lys(1)stop (84 times), Leu(2)Leu(1)stop (68 times), Leu(2)Lys(1)stop (59 times) and Arg(2)Lys(1)stop (57 times). The occurrence of the hydrophobic amino acid leucine in these contexts hints at the second possible mechanism of proteinlipid interaction provided by lipophilic residues.
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Proteinlipid interactions of randomly expressed GST-fusion peptides
To investigate systematically the binding properties of randomly expressed peptides to phospholipids we constructed an expression library from genomic yeast DNA (for details, see Material and methods). After partial digestion of genomic DNA with the restriction endonuclease Sau3AI only fragments longer than 1000 base pairs were cloned into the BamHI site of the plasmid pEG(KT). This brings the DNA fragments in frame with the GST gene encoded by the vector. Since there is no preference for any given ORF, <15% of expressed fusion peptides should be a part of the naturally expressed yeast proteins. (For the final analysis of binding data we excluded these naturally occurring protein fragments.) The complete set of library plasmids was transformed into the S.cerevisiae strain BJ5459. To test the GST-fusion proteins for phospholipid binding we inoculated 3800 independent yeast clones. The expressed fusion proteins were immobilized on glutathioneSepharose and subjected to a phospholipid-binding assay as described in Materials and methods.
In this screen approximately 80 yeast clones were identified to express GST-fusion proteins which bind phospholipid vesicles. From these clones, plasmids were extracted, amplified, retransformed into yeast and binding properties were verified, indicating that lipid binding is plasmid dependent. Immobilized proteins were subjected to PAGE analysis. The extent of purification usually exceeded 95%. Subsequent DNA-sequencing analysis revealed that the plasmids identified encoded 20 fragments of known yeast proteins and 54 different peptide fragments not expressed naturally in yeast. From the set of peptide fragments, 31 were found to be Lys/Arg-rich and showed at least one Lys/Arg residue among the last five C-terminal amino acid residues or more than two Lys/Arg residues in the last 20 C-terminal amino acid residues (Table II) which corresponds to 57% of found sequences. If the subset of Lys/Arg-rich fragments is analysed for the appearance of the basic residues Lys or Arg at distinct positions, both amino acids are over-represented in the positions (1) and (2) and under-represented at positions (3), (4) and (5) (Table III
). Our results clearly demonstrate the preponderance of Lys/Arg-rich fragments among randomly expressed peptides which bind phospholipids in vitro. Most efficient binding is obtained with basic residues located close to the C-terminus.
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To prove this hypothesis we investigated the C-terminal region of Sip18p, a yeast protein, which shows high sequence similarity to the phospholipid-binding peptide no. 9 from Table II. Computational analysis reveals 70% identity and 90% similarity for the immediate C-terminal region. Sip18p is involved in the osmotic stress response whose regulation is known to involve phosphoinositides. SIP18 is not essential for the viability of S.cerevisiae and expression was demonstrated on the mRNA level (Miralles and Serrano, 1995
). The SIP18 ORF was amplified by PCR from genomic DNA, verified by DNA sequencing and cloned into the expression plasmid pEG(KT). Since Sip18p is negatively regulated by proteinase A, the protease-deficient yeast strain BJ5459 was transformed. It could be shown that Sip18p strongly binds to phospholipid vesicles (Figure 2
).
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Discussion |
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To investigate systematically the binding properties of C-terminal peptides we used a yeast genomic expression library. GST-fusion proteins were randomly expressed in S.cerevisiae. Single colonies were grown, cells were lysed and immobilized GST-fusion proteins were tested for binding to phosphoinositide-containing vesicles. Among the 3800 clones tested we identified 54 peptide sequences which are not derived from known yeast ORFs. Nearly 60% of these naturally not translated sequences are found to encode Lys/Arg-rich peptides. A comparison of all binding sequences suggested the hypothesis that paired basic amino acid residues close to the C-termini of proteins are sufficient for binding negatively charged phospholipids. This fact should be taken into account if peptides were designed or if lipid-binding domains of proteins should be mapped. Mutational analysis of rabphilin3a identified one peptide from the C-terminal region of the C2B domain that specifically inhibited secretion from permeabilized chromaffin cells and binding of rabphilin3a to phospholipid vesicles. This peptide showed the C-terminal sequence [..KDKKIstop] (Chung et al., 1998).
Lipid binding seems not only to depend on the number of Lys/Arg residues but also on their position along the polypeptide chain. Lys/Arg residues at C-terminal positions (1) and (2) were over-represented among the identified clones and therefore we suppose that these constructs allow proteins to attach their C-termini to membrane components such as negatively charged phospholipids. We further investigated the lipid-binding behaviour of the yeast protein Sip18p which shows high sequence similarity to the phospholipid-binding peptide no. 9 from Table II and which is characterized by a double lysine motif at the C-terminus. The wild-type protein showed binding to phospholipid vesicles in vitro, whereas deletion of the C-terminal lysine residues abolished lipid binding. The localization of GFP-fusion constructs of Sip18p in vivo was also found to be dependent on the C-terminal lysine residues (data not shown). In the case of several proteins it was shown that C-terminal basic residues are necessary for proper cellular function. It was suggested that the C-terminal basic residues of bacterial galactosyltransferases may be responsible for anchoring the protein to the membrane through an ionic interaction with negatively charged phospholipids (Wakarchuk et al., 1998
). Melittin, a small lytic polypeptide from honeybee venom, has a profoundly disruptive effect on membranes which is abolished by selective replacement of the C-terminal basic amino acids (van Veen et al., 1995
). In the case of human ras it was recently shown that the polybasic domain influences plasma membrane accumulation (Apolloni et al., 2000
).
Therefore, we conclude that a Lys/Arg terminus confers the possibility of phospholipid interactions and this fact could contribute to explain the preference for lysine in natural protein termini among a variety of species. We suggest that paired basic residues in the immediate C-terminal regions should be taken into account if lipid-binding properties of proteins are considered both in vitro and in vivo.
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Notes |
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Acknowledgments |
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References |
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Arkov,A.L., Korolev,S.V. and Kisselev,L.L. (1995) Nucleic Acid Res., 23, 47124716.[Abstract]
Baraldi,E., Djinovic Carugo,K., Hyvönen,M., Lo Surdo,P., Riley,A.M., Potter,B.V.L., O'Brien,R., Ladbury,J.E. and Saraste,M. (1999) Struct. Fold. Design, 7, 449460.
Barret,C., Roy,C., Montcourrier,P., Mangeat,P. and Niggli,V. (2000) J. Cell Biol., 151, 10671079.
Berezovsky,I., Kilosanidze,G.T., Tumanyan,V.G. and Kisselev,L. (1997) FEBS Lett., 404, 140142.[CrossRef][ISI][Medline]
Berezovsky,I., Kilosanidze,G.T., Tumanyan,V.G. and Kisselev,L.L. (1999) Protein Eng., 12, 2330.
Bertram,G., Innes,S., Minella,O., Richardson,J.P. and Stansfield,I. (2001) Microbiology, 147, 255269.
Bjornsson,A., Mottagui-Tabar,S. and Isaksson,L.A. (1996) EMBO J., 15, 16961704.[Abstract]
Bonetti,B., Fu,L., Moon,J. and Bedwell,D.M. (1995) J. Mol. Biol., 251, 334345.[CrossRef][ISI][Medline]
Bottomley,M.J., Salim,K. and Panayotou,G. (1998) Biochim. Biophys. Acta, 1436, 165183.[ISI][Medline]
Burd,C.G. and Emr,S.D. (1998) Mol. Cell, 2, 157162.[ISI][Medline]
Christopher,J.A. and Baldwin,T.O. (1996) J. Mol. Biol., 257, 175187.[CrossRef][ISI][Medline]
Chung,S.-H., Song,W.-J., Kim,K., Bednarski,J.J., Chen,J., Prestwich,G.D. and Holz,R.W. (1998) J. Biol. Chem., 273, 1024010240.
Fruman,D.A., Rameh,L.E. and Cantley,L.C. (1999) Cell, 97, 817820.[ISI][Medline]
Gaidarov,I. and Keen,J.H. (1999) J. Cell Biol., 146, 755764.
Gaullier,J.-M., Simonsen,A., D'Arrigo,A., Bremnes,B., Aasland,R. and Stenmark,H. (1998) Nature, 394, 432433.[CrossRef][ISI][Medline]
Hamada,K., Shimizu,T., Matsui,T., Tsukita,S., Tsukita,S. and Hakoshima,T. (2000) EMBO J., 19, 44494462.
Harlan,J.E., Hajduk,P.J., Yoon,H.S. and Fesik,S.W. (1994) Nature, 371, 168170.[CrossRef][ISI][Medline]
Hurley,J.H. and Misra,S. (2000) Annu. Rev. Biomol. Struct., 29, 4979.[CrossRef][ISI][Medline]
Isakoff,S.J., Cardozo,T., Andreev,J., Li,Z., Ferguson,K.M., Abagyan,R., Lemmon,M.A., Aronheim,A. and Skolnik,E.Y. (1998) EMBO J., 17, 53745387.
Janmey,P.A., Lamb,J., Allen,P.G. and Matsudaira,P.T. (1992) J. Biol. Chem., 267, 1181811823.
Jorgensen,E.M., Hartwieg,E., Schuske,K., Nonet,M.L., Jin,Y. and Horvitz,H.R. (1995) Nature, 378, 196199.[CrossRef][ISI][Medline]
Kang,H., Freund,C., Duke-Cohan,J.S., Musacchio,A., Wagner,G. and Rudd,C.E. (2000) EMBO J., 19, 28892899.
Kirsch,C., Wetzker,R. and Klinger,R. (2001) Biochem. Biophys. Res. Comm., 282, 691696.[CrossRef][ISI][Medline]
Martin,T.F.J. (1998) Annu. Rev. Cell Dev. Biol., 14, 231264.[CrossRef][ISI][Medline]
Mayer,A., Scheglmann,D., Dove,S., Glatz,A., Wickner,W. and Haas,A. (2000) Mol. Biol. Cell, 11, 807817.
McCaughan,K.K., Brown,C.M., Dalphin,M.E., Berry,M.J. and Tate,W.P. (1995) Proc. Natl Acad. Sci. USA, 92, 54315435.[Abstract]
Miralles,V.J. and Serrano,R. (1995) Mol. Microbiol., 17, 653662.[ISI][Medline]
Mitchell,D.A., Marshall,T.K. and Deschened,R.J. (1993) Yeast, 9, 715723.[ISI][Medline]
Mottagui-Tabar,S., Tuite,M.F. and Isaksson,L.A. (1998) Eur. J. Biochem., 257, 249254.[Abstract]
Prockop,D.J. and Kivirikko,K.I. (1995) Annu. Rev. Biochem., 64, 403434.[CrossRef][ISI][Medline]
Sciorra,V.A., Rudge,S.A., Prestwich,G.D., Frohman,M.A., Engebrecht,J.A. and Morris,A.J. (1999) EMBO J., 20, 59115921.[CrossRef]
Soom,M., Schönherr,R., Kubo,Y., Kirsch,C., Klinger,R. and Heinemann,S.H. (2001) FEBS Lett., 490, 4953.[CrossRef][ISI][Medline]
van Veen,M., Georgiou,G.N., Drake,A.F. and Cherry,R.J. (1995) Biochem. J., 305, 785790.[ISI][Medline]
Wakarchuk,W.W., Cunningham,A., Watson,D.C. and Young,N.M. (1998) Protein Eng., 11, 295302.[Abstract]
Xue,H.-W., Pical,C., Brearley,C., Elge,S. and Müller-Röber,B. (1999) J. Biol. Chem., 274, 57385745.
Yagisawa,H., Sakuma,K., Paterson,H.F., Cheung,R., Allen,V., Hirata,H., Watanabe,Y., Hirata,M., Williams,R.L. and Katan,M. (1998) J. Biol. Chem., 273, 417424.
Received May 25, 2001; revised January 21, 2002; accepted February 12, 2002.