Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.     

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.     

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.     

Structure and properties of calphobindin II, an anticoagulant protein from human placenta

The structure of human placental calphobindin-II (CPB-II) was investigated by amino acid composition and amino acid sequence analyses of peptides generated by protease digestion of the protein. The 45 peptides obtained from the lysyl endopeptidase digest of CPB-II, and the amino-terminal peptide prepared from its tryptic digest, were analyzed, and they accounted for over 98% of total amino acids of CPB-II. The structure of CPB-II determined by protein sequencing was identical to that previously predicted from its cDNA sequence (Iwasaki, A. et al. (1989) J. Biochem. 106, 43-49), except for the amino terminus. Since the amino terminus of CPB-II was blocked to Edman degradation, fast-atom-bombardment mass spectrometric analysis was used to demonstrate that the amino-terminal residue was acetyl-alanine. The carboxyl-terminal residue of CPB-II was identified as aspartic acid by the hydrazinolytic procedure. Calcium-binding studies indicated that 1 mol of CPB II binds 1 mol of calcium in the absence of phospholipid and 8 mol of calcium in the presence of phospholipid.     

Protein structural requirements and properties of membrane binding by gamma-carboxyglutamic acid-containing plasma proteins and peptides

The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.     

Canine hepatic lysosomal copper protein: identification as metallothionein

We studied the amino acid sequence of canine hepatic lysosomal copper protein obtained from Bedlington terriers affected by inherited copper toxicosis. The primary structure was determined by manual Edman degradations and carboxypeptidase Y digestions of peptides generated by cleavage of the S-carboxyamidomethylated and S-aminoethylated protein with trypsin. Although the amino terminus was blocked and heterogeneous, the protein showed extensive sequence homology to mammalian metallothioneins. In particular, all cysteinyl residues were conserved, in agreement with their function as metal ligands. The microheterogeneity observed in the amino-terminal part of the molecule indicated the presence of two isoforms in canine liver like those found in most other mammals studied so far.     

Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene

A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed

Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues.     

Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein

Protein synthesis initiation factor 4D (eIF-4D) from mammalian cells contains the post-translationally modified lysine derivative hypusine. A highly purified preparation of the protein from rabbit reticulocytes was subjected to chemical and enzymatic cleavage, and a large number of overlapping peptides were resolved by high performance liquid chromatography and sequenced. Two mixed 14-base DNA probes were synthesized based on suitable amino acid sequences and were used to screen a human cDNA library in lambda gt11. A cDNA insert containing eIF-4D encoding sequences was identified and a 558-base pair EcoRI-PstI fragment was sequenced. Northern blot hybridization of HeLa cell RNA shows a single size class (1.2 kilobase) of mRNA. The DNA encodes a protein comprising 154 residues with a mass of 16,703 daltons. Human eIF-4D matches all of the rabbit peptides sequenced, extending from residue 9 to 154 except for Cys-129 which is Ser in the rabbit protein. The residue modified to hypusine is identified as Lys-50 and the amino terminus is blocked. eIF-4D possesses rather little secondary structure in the amino-terminal two-thirds of the protein, but the carboxyl-terminal third is rich in alpha helices.     

Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase.

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.     

Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides

We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates that permit continuous measurement of the enzyme activity, even in crude tissue extracts.     

Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.     

Novel thioester reagents afford efficient and specific S-acylation of unprotected peptides under mild conditions in aqueous solution.

S-acylated peptides have many potential uses for elucidating the biophysical, structural and other properties of the numerous S-acylated proteins of mammalian cells. However, with the currently available reagents, preparation of specifically S-acylated derivatives of peptides is generally laborious or simply unfeasible. We here show that novel, easily preparable aryl and alkyl thioester derivatives of palmitic acid can mediate S-acylation of peptides corresponding to physiologically S-acylated sequences from the proteins p56(lck) and H-ras and the Po glycoprotein of peripheral myelin, with high selectivity for cysteine over other amino acid functional groups (including hydroxyl and both alpha- and epsilon-amino residues), and with much greater efficiency than is obtained using acyl-coenzyme A derivatives. Efficient and selective S-acylation can be accomplished under very mild conditions in aqueous systems containing lipid vesicles or detergent micelles, or in homogenous aqueous/acetonitrile mixtures. Using these novel thioesterifying reagents, we confirm previous suggestions that the N-terminal cysteine residue of Hedgehog proteins can exhibit rapid, uncatalyzed S-to-N acyl transfer following S-acylation to produce the N-palmitoylated amino terminus found in the mature protein. By contrast, we demonstrate that spontaneous S-to-N acyl transfer from the cysteine to the terminal glycine residue in the amino-terminal peptide of G(alphas) is far less rapid and is likely too slow to explain the physiological N-palmitoylation of the amino terminus of this protein.     

Use of the pH Memory Effect in Lyophilized Proteins to Achieve Preferential Methylation of α-Amino Groups

It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the α-amino groups were found to be either preferentially or selectively trimethylated. Reaction with ¹³C-labeled iodomethane permitted detection and identification of individual trimethylated α-amino groups by ¹³C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free α-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated α-amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential ¹³C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus.     

Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme

Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.     

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).     

The amino acid sequence of avian thymic hormone, a parvalbumin

The complete amino acid sequence of 'avian thymic hormone' (ATH), a protein from thymus tissue that appears to promote immune maturation in chicken bone marrow cells in culture, is presented. The sequence was obtained from sequences of ATH peptides isolated by HPLC after tryptic, chymotryptic, peptic or S aureus V8 protease digestions. The protein is a parvalbumin consisting of 108 residues with a blocked amino terminus, a single cysteine, tyrosine, proline and arginine and no histidine, methionine or tryptophan. This is the first amino acid sequence of a parvalbumin which is not derived from muscle tissue.     

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.     

Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation

The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated with light, the 25- and 27-kDa forms of the light-harvesting chlorophyll a/b protein (LHC II) were phosphorylated on their amino termini. Treatment of the membranes with proteinase K or thermolysin released phosphopeptides which were purified by ferric ion affinity chromatography and reverse phase high performance liquid chromatography. Sequencing of the phosphopeptides was performed with tandem quadrupole mass spectrometry. Three different phosphopeptides Ac-RKTAGKPKT, Ac-RKTAGKPKN, and Ac-RKSAGKPKN originating from class I LHC II were examined after release by thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, originating from class II LHC II was examined after release by proteinase K. Each of the four LHC II phosphopeptides was derived from the amino terminus of a distinct protein. Peptides were acetylated at their amino-terminal arginine and were phosphorylated on either threonine or serine in the third position. We conclude that proteolytic processing of pre-LHC II occurs at a conserved methionyl-arginyl bond and is followed by amino-terminal acetylation of the arginine and nearby phosphorylation of the mature LHC II. Eight different peptides were synthesized in acetylated and nonacetylated forms as substrates for the thylakoid membrane kinase. From a comparison of the kinetics of phosphate incorporation into the peptides, we conclude that basic residues on both sides of the phosphorylation site are important for enzyme recognition. Acetylation of the amino terminus is not required for phosphorylation.