Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.


Introduction
Protein N-myristoylation is the attachment of myristic acid, a 14-carbon saturated fatty acid, to the N-terminal Gly of proteins [1][2][3][4][5]. This modification is one of the major forms of lipid modification that occurs on eukaryotic and viral proteins. It is estimated that approximately 0.5-1.5% of eukaryotic proteins are N-myristoylated. In general, myristic acid is cotranslationally attached to the N-terminal Gly residue after removal of the initiating Met. In addition to cotranslational protein N-myristoylation, it has been established that posttranslational N-myristoylation can also occur on many caspase-cleavage products in apoptotic cells [6][7][8]. Both cotranslational and posttranslational N-myristoylation are catalyzed by N-myristoyltransferase (NMT), a member of the GCN5-related N-acetyltransferase superfamily of proteins [9]. The precise substrate specificity of this enzyme has been characterized using purified enzyme and synthetic peptide substrates [10,11]. The requirement for Gly at the N-terminus is absolute and no other amino acid can take its place.
Many N-myristoylated proteins play key roles in regulating cellular structure and function. They include proteins involved in a wide variety of cellular signal transduction pathways, such as protein kinases, phosphatases, guanine nucleotide binding proteins, Ca 2+ binding proteins, and cytoskeletal regulatory proteins [1][2][3][4][5]. In addition to proteins involved in cellular signal transduction pathways, recent studies have revealed that protein N-myristoylation occurs on many disease-related proteins [12][13][14][15][16]. However, comprehensive identification of human Nmyristoylated proteins has not been achieved because of the lack of a simple and easy strategy to detect protein N-myristoylation.
Recent progress in chemical biology has made novel approaches available for the study of protein N-myristoylation by taking advantage of bioorthogonal reactions [17,18]. The advantage of these approaches is that they are non-radioactive, have short detection times and a high degree of sensitivity in comparison with traditional radiolabeling. In fact, metabolic labeling of cellular proteins with bioorthogonal myristic acid analogues and subsequent ligation with secondary reporters enables visualization, enrichment, and MS-based identification of N-myristoylated proteins [19,20]. In addition to these protein-based strategies, human Nmyristoylated proteins could be identified by cell-free and cellular metabolic labeling using human cDNA clones. In our previous study, to identify novel human N-myristoylated proteins, the susceptibility of human cDNA clones from human cDNA resources to protein N-myristoylation was evaluated by metabolic labeling and mass spectrometric analyses of proteins expressed using an insect cell-free protein synthesis system [21]. As a result, the products of 18 out of~2,000 cDNA clones (Kazusa ORFeome project human cDNA clones) were found to be novel N-myristoylated proteins that had not been reported previously. These results indicated that this strategy is useful for the identification of human N-myristoylated proteins from human cDNA resources.
In this study, to identify physiologically important human N-myristoylated proteins, cellfree-and cellular metabolic labeling experiments coupled with bioinformatic prediction systems for protein N-myristoylation were performed using 4,369 Kazusa ORFeome project human cDNA clones.
Prediction of protein N-myristoylation using prediction programs Two public WWW-server-based prediction programs for protein N-myristoylation, MYR Predictor (http://mendel.imp.ac.at/myristate/SUPLpredictor.htm) [22] and Myristoylator (http:// www.expasy.org/tools/myristoylator/) [23], were used for the prediction of protein N-myristoylation. The entire amino acid sequences deduced from the nucleotide sequences of the ORFs were used as the query.

Plasmid construction
Nucleotide sequences of oligonucleotide primers used for plasmid construction are summarized in S1 Table. Plasmid pTD1 (Shimadzu) was used for the insect cell-free protein synthesis system [24]. Plasmids pcDNA3-tActin-FLAG, pcDNA3-tActinG2A-FLAG, pTD1-tActin-FLAG, pTD1-tActinG2A-FLAG and pcDNA3-FLAG were constructed as described previously [7,25]. Construction of the expression vector pTD1-Δ10aa-tActin-Flag for screening N-myristoylated proteins is summarized in S2 Table. pTD1 plasmids containing the cDNAs coding for the tActin fusion proteins with N-terminal ten amino acid sequences of the ORF of the KOP cDNA clones at the N-terminus were constructed as follows. The two oligonucleotides, 5'-ATCNNNNNNNNNN NNNNNNNNNNNNNNNG-3' (sense strand) and 5'-AATTCNNNNNNNNNNNNNNNNNN NNNNNNNGAT-3' (antisense strand), coding for the N-terminal 10 amino acid sequence of the ORF of the KOP cDNA clones were annealed. The annealed dsDNAs were individually ligated into EcoRV-EcoRI sites of the pTD1-Δ10aa-tActin-FLAG vector. The resulting plasmids were designated as pTD1-NNNNN-tActin-FLAG, where NNNNN indicates the number of the product ID of the KOP cDNA clones. The construction of the pTD1 or pcDNA3 plasmids including fulllength KOP cDNA clones are summarized in S2 Table. In vitro transcription and translation reaction mRNAs encoding the cDNAs were prepared using a T7-scribe standard RNA IVT kit (CELL-SCRIPT) in accordance with the manufacturer's instructions. The synthesized mRNAs were purified by phenol-chloroform extraction and ethanol precipitation before use in the translation reaction.

Cell-free protein synthesis
The translation reaction was performed using an insect cell-free protein synthesis system (Shimadzu) in the presence of [ 3 H]leucine or [ 3 H]myristic acid as described previously [25]. The mixture (composed of 6.2 μL insect cell lysate, 3.7 μL reaction buffer, 0.5 μL 4 mM methionine, 1

Transfection of cells
HEK293T (a human embryonic kidney cell line) cells or COS-1 (simian virus 40-transformed African green monkey kidney cell line, American Type Culture Collection) cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL [Palo Alto, CA, USA]) supplemented with 10% fetal calf serum (FCS; Gibco BRL). Cells (2 × 10 5 ) were plated onto 35-mm diameter dishes 1 day before transfection. pcDNA3 constructs (2 μg) containing cDNAs encoding FLAG-tagged proteins were used to transfect the cells in each plate along with 2.5 μL Lipofectamine LTX and 2 μL Plus reagent in 1 mL serum-free medium. After incubation for 5 h at 37°C, the cells were re-fed with serum-containing medium and incubated again at 37°C for appropriate periods.

SDS-PAGE and fluorography
The radiolabeled proteins were resolved by 12.5% SDS-PAGE, then the gel was fixed and soaked in ENLIGHTNING (PerkinElmer) for 20 min. Thereafter, the gel was dried under vacuum and exposed to X-ray film for an appropriate period.

Western blotting
Proteins were resolved by 12.5% SDS-PAGE and then transferred to an Immobilon-P transfer membrane. After blocking with non-fat milk, the membrane was probed with a primary antibody, as described previously [27]. Immunoreactive proteins were detected specifically by incubation with protein G-HRP conjugate. The membrane was developed using ECL Prime western blotting detection reagent or ImmunoStar LD and detected using a MicroChemi Chemiluminescence Imaging System. The blots were quantified by densitometry using the software TotalLab Quant.

Immunofluorescence analysis and fluorescence microscopy
Immunofluorescence analysis of transfected cells was performed 24 h after transfection [28]. After staining with Hoechst 33342 and MitoTracker Red, the cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS. The permeabilized cells were incubated with anti-SAMM50 antibody (HPA034537) in DPBS for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were incubated with anti-Rabbit IgG-FITC antibody for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were observed using a Leica AF7000 fluorescence microscope (Leica, Solmser, Germany). Quantitative analysis of the mitochondrial localization of SAMM50 was performed by fluorescence microscopic observation of 50 immunofluorescence-positive (transfected) cells. The extent of mitochondrial localization was expressed as a percentage of the number of cells in which selective localization to mitochondria, localization to both mitochondria and cytoplasm, and selective localization to the cytoplasm was observed against the total number of transfected cells. Data are expressed as mean ± SD for 5 independent experiments.

Statistic analysis
Statistical analysis was carried out using two-tailed t test (Microsoft Excel; Microsoft). The means of two distributions were considered significantly different if p < 0.05.

Results
Selection of candidate cDNA clones encoding human N-myristoylated proteins from a cDNA resource In order to search for human N-myristoylated proteins, 339 cDNA clones with N-terminal Met-Gly motifs were extracted from 4,369 KOP (Kazusa ORFeome project) human cDNA clones (FXC01948~FXC23818) (Fig 1A). After applying the N-terminal sequence of the products of these 339 cDNA clones to two protein N-myristoylation prediction programs, The MYR Predictor and Myristoylator, 90 positively selected cDNA clones were indentified (S3 Table). From these cDNA clones, 53 clones coding for known N-myristoylated proteins identified by database searches were removed, and 37 cDNA clones were selected as potential candidates for human N-myristoylated proteins ( Fig 1A). The samples analyzed are listed in S4 Table. The susceptibility of human cDNA clones to protein N-myristoylation was evaluated using fusion proteins in which the N-terminal 10 amino acid residues were fused to an epitopetagged model protein, tActin ( Fig 1B). In this experiment, the fusion proteins were synthesized using an insect cell-free protein synthesis system in the presence of [ 3 H]leucine or [ 3 H]myristic acid, and then analyzed by SDS-PAGE and fluorography. As shown in the upper panels of Fig  1C, most of the cDNA clones were expressed, as determined by the incorporation of [ 3 H]leucine. In contrast, the incorporation of [ 3 H]myristic acid was observed for 19 out of 37 clones, as shown in the middle panels of Fig 1C. The results of the prediction for protein N-myristoylation using two prediction programs, The MYR Predictor and Myristoylator, are shown in the lower panels of Fig 1C. Analyses of protein N-myristoylation occurring in in vitro synthesized fulllength cDNA products To determine whether the experimental results obtained with the tActin fusion proteins were consistent with those with the full-length cDNA products, metabolic labeling experiments were performed using the 19 full-length cDNAs in which incorporation of [ 3 H]myristic acid was observed with the tActin fusion proteins. The samples analyzed are listed in S5 Table. As shown in the upper panels of Fig  Analyses of protein N-myristoylation occurring in full-length cDNA products expressed in transfected human cells To determine whether results obtained by in vitro metabolic labeling in the insect cell-free protein synthesis system reflected the in vivo behavior of the cDNA products, metabolic labeling in transfected HEK293T (a human embryonic kidney cell line) cells was performed using the 19 full-length cDNAs analyzed in the insect cell-free protein synthesis system (S5 Table). In contrast to the insect cell-free protein synthesis system, not all the cDNA clones were expressed in transfected HEK293T cells. Protein synthesis was observed for 11 cDNA clones, as determined by the western blotting analysis using an anti-FLAG antibody. Protein expression was  Characteristics of the gene products of 13 human cDNA clones found to be N-myristoylated in this study The characteristics of the gene products of 13 human cDNA clones found to be N-myristoylated in this study are summarized in Table 1.
The 13 N-myristoylated proteins found in this study included key components in various cellular signal transduction pathways such as a protein kinase, E3-ubiquitin ligase component, cancer-related protein, apoptosis-related protein, but also integral transmembrane proteins that play critical roles in cellular functions.
Protein N-myristoylation strongly affects the mitochondrial targeting of SAMM50, an outer membrane protein of mitochondria In order to determine the physiological importance of protein N-myristoylation occurring on human N-myristoylated proteins found in this study, the role of protein N-myristoylation on the intracellular localization of SAMM50, an outer membrane protein of mitochondria, was investigated. SAMM50 is a central component of the sorting and assembly machinery (SAM) necessary for the assembly of β-barrel proteins in the mitochondrial outer membrane [31][32][33]. Interspecies alignments revealed that the N-terminal N-myristoylation motif were highly conserved among vertebrates, as shown in Fig 4A. For this analysis, a non-N-myristoylated G2A mutant, in which Gly2 was replaced with Ala, was constructed and its intracellular localization was compared with that of wild-type protein.
As shown in Fig 4B, efficient expression of wild-type and G2A mutant of SAMM50 tagged with FLAG-tag was observed as determined by western blotting using an anti-SAMM50 antibody. In contrast, protein expression was not detected by western blotting using an anti-FLAG antibody, as described previously. Metabolic labeling with [ 3 H]myristic acid revealed that efficient incorporation of [ 3 H]myristic acid was observed in wild-type SAMM50, but the incorporation was completely inhibited by replacing Gly2 with Ala. The results of immunofluorescence staining of COS-1 (simian virus 40-transformed African green monkey kidney cell line) cells transfected with these cDNAs revealed that protein N-myristoylation strongly affected the intracellular localization of SAMM50, as shown in Fig 4C. Immunofluorescence staining with the anti-SAMM50 antibody coupled with MitoTracker staining revealed that N-myristoylated SAMM50-FLAG exclusively localized to mitochondria, whereas the nonmyristoylated G2A mutant localized mainly to the cytoplasm. In order to confirm the role of protein N-myristoylation in mitochondrial targeting of SAMM50, quantitative analysis of the intracellular localization of SAMM50-FLAG and SAMM50-G2A-FLAG expressed in COS-1 cells was performed by fluorescence microscopic observation. As a result, significant differences in intracellular localization were observed with these two proteins, as shown in Fig 4D. In the case of SAMM50-FLAG, most of the expressed proteins were specifically localized to the mitochondria. In contrast, as for SAMM50-G2A-FLAG, cytoplasmic localization (cytoplasm or mitochondria/cytoplasm) was observed in~70% of transfected cells (Fig 4D). These results clearly indicated that protein N-myristoylation plays critical roles in the proper targeting of SAMM50 to mitochondria.

Endogenous SAMM50 expressed in mammalian cells is N-myristoylated
To determine whether protein N-myristoylation was observed on endogenous SAMM50 expressed in mammalian cells, metabolic labeling of endogenous SAMM50 expressed in COS-1 cells with [ 3 H]myristic acid was performed. As shown in Fig 5A, lane 2, expression of endogenous SAMM50 was detected by western blotting analysis using an anti-SAMM50 antibody.
In this case, the molecular size of endogenous SAMM50 was similar to that of Tag

Discussion
Protein N-myristoylation is one of the major forms of lipid modification that occurs in human cells. However, because of the lack of a simple and easy strategy to detect protein N-myristoylation, comprehensive identification of human N-myristoylated proteins has not been achieved.
Recently, a novel approach based on chemical biology has become available for the study of protein N-myristoylation by taking advantage of bioorthogonal reactions [17][18][19][20]. For example, it was recently reported that metabolic labeling of cellular proteins with bioorthogonal myristic acid analogues and subsequent ligation with secondary reporters followed by enrichment and MS-based analysis successfully identified~100 human N-myristoylated proteins expressed in HeLa cells [19]. Thus, this approach is extremely useful in detecting the N-myristoylated proteins expressed in intact cells. In this approach, relatively abundant constitutively expressed N-myristoylated proteins in the cells can be detected. However, for some cellular proteins, the amount of protein produced is lower than the detection limit of MS analysis. In addition, it is well known that only a limited sets of genes are expressed in particular cells. Hence, these approaches may not be suitable for the comprehensive identification of human N-myristoylated proteins expressed in whole human cells or tissues. Therefore, other approaches to identify human N-myristoylated proteins are required to fulfill the comprehensive identification of human N-myristoylated proteins. It has been shown that protein N-myristoylation could be detected by metabolic labeling in various cell-free protein synthesis systems using cDNA clones [34][35][36]. In fact, we have demonstrated that a newly developed cell-free protein synthesis system (Transdirect insect cell) derived from insect cells [37] can be used for in vitro metabolic labeling assays, and that the metabolic labeling in this insect cell-free system is a simple and sensitive method to detect protein N-myristoylation [35]. In our previous study, to identify novel human N-myristoylated proteins, the susceptibility of human cDNA clones from human cDNA resource to protein Nmyristoylation was evaluated by metabolic labeling and mass spectrometric analyses of proteins expressed using an insect cell-free protein synthesis system [21]. For this analysis, 141 cDNA clones with an N-terminal Met-Gly motif were selected as potential candidates from 2,000 KOP (Kazusa ORFeome project) human cDNA clones. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins, in which the N-terminal 10 amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation on the gene product of the full-length cDNA was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected COS-1 cells. As a result, the products of 27 out of~2,000 cDNA clones were found to be N-myristoylated. Among them, 18 proteins were novel N-myristoylated proteins that had not been reported previously. These results indicated that this strategy is useful for the identification of human N-myristoylated proteins from human cDNA resources. However, in this strategy, the efficiency of the detection of N-myristoylated protein from tested cDNA clones was low (27 out of 141 tested proteins). Therefore, in the present study, in order to increase the detection efficiency, cell-free-and cellular metabolic labeling experiments coupled with bioinformatic prediction systems for protein N-myristoylation were performed to identify physiologically important human N-myristoylated proteins. A bioinformatic approach is a powerful strategy to perform comprehensive identification of N-myristoylated proteins [22,23,38,39]. Two prediction programs, The MYR Predictor and Myristoylator are available as public WWW-servers [22,23]. In the previous study, to evaluate the reliability of these prediction programs, experimental results of metabolic labeling with 141 proteins with a Met-Gly sequence at the N-terminus were compared with the results of the prediction obtained by the two prediction programs [21]. As a result, it was revealed that the reliability of The MYR Predictor was high; however, there were also a considerable number of false-negative predictions. In contrast, the Myristoylator predicted many more N-myristoylated proteins, but there were many false-positive predictions. When the positively predicted proteins by either of the two prediction programs were combined, most of the N-myristoylated proteins were predicted correctly. Therefore, in the present study, the proteins predicted to be N-myristoylated by either of the two prediction programs were used as candidates for human N-myristoylated proteins.
In order to identify physiologically important human N-myristoylated proteins, the susceptibility of the human cDNA clones in human cDNA resource to protein N-myristoylation was evaluated by metabolic labeling in a cell-free protein synthesis system coupled with bioinformatic prediction systems. For this analysis, 90 cDNA clones with an N-terminal Met-Gly motif predicted to be N-myristoylated by two bioinformatic N-myristoylation prediction systems were selected from~4,400 Kazusa ORFeome project (KOP) human cDNA clones (FXC01948~FXC23818) (S1 Table). After database searches for known N-myristoylated proteins, 37 cDNA clones were selected as potential candidates for human N-myristoylated proteins (S2 Table). The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins, in which the N-terminal 10 amino acid residues were fused to an epitope-tagged model protein (Fig 1). Then, protein N-myristoylation on the gene product of the full-length cDNA was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells (Figs 2 and 3).
As for protein N-myristoylation occurring on integral membrane proteins, we have recently demonstrated that protein Lunapark, a double-spanning integral membrane protein of the ER, is N-myristoylated and the N-myristoylation of protein Lunapark strongly affected ER morphological changes induced by this protein [40]. Thus, it seems likely that protein N-myristoylation plays critical role in the function of integral membrane proteins. However, so far, very few integral membrane proteins has been found to be N-myristoylated. SAMM50, one of the human N-myristoylated proteins found in this study, is an integral membrane protein of the outer membrane of mitochondria. Therefore, we focused our attention on the protein N-myristoylation occurring on SAMM50. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50 by immunofluorescence analysis revealed that protein Nmyristoylation strongly affected the subcellular localization of SAMM50. N-myristoylated SAMM50-FLAG was found to localize exclusively to mitochondria, whereas a non-myristoylated G2A mutant localized mainly to the cytoplasm (Fig 4C and 4D). Furthermore, immunoprecipitiation analysis of [ 3 H]myristic acid-labeled cellular proteins revealed that endogenous SAMM50 is N-myristoylated (Fig 5). Thus, it was demonstrated that SAMM50 expressed in intact cells is indeed N-myristoylated, and this modification plays a critical role in the intracellular localization of this protein. In eukaryotic cellular proteins, only very few integral membrane proteins have been demonstrated to be N-myristoylated. One well-characterized example of an N-myristoylated integral membrane protein in eukaryotes is mammalian NADH-cytochrome b(5) reductase (b5R). This protein is a single-spanning membrane protein with N-exo/C-cyto orientation, and it is dually targeted to the ER and mitochondrial outer membranes [41,42]. In b5R, protein N-myristoylation is required for targeting to the mitochondria, with a non-myristoylated mutant exclusively localized to the ER [43]. It was further revealed that protein N-myristoylation interferes with the interaction of the nascent chain with the signal recognition particle, so that a portion of the nascent chains escape from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane [44]. SAMM50 is a β-barrel protein that resides within the outer membrane of mitochondria [32,33]. Unlike most of the transmembrane proteins that are anchored in the lipid bilayer via membrane spanning α-helices, β-barrel proteins transverse the membrane by interconnected β-strands [45]. It was revealed that all the mitochondrial outer membrane βbarrel proteins carry internal non-cleavable targeting and sorting signals [46]. Because SAMM50 does not contain a hydrophobic sequence that interacts with the signal recognition particle, it is probable that protein N-myristoylation of SAMM50 does not affect the interaction of the nascent polypeptide with the signal recognition particle. In fact, most of the non-myristoylated G2A-mutant of SAMM50 did not localize to ER but was found to localize to the cytoplasm (Fig 4C). The molecular mechanism by which protein N-myristoylation affects the mitochondrial targeting of SAMM50 is not clear. β-barrel proteins do not enter the outer mitochondrial membrane from the cytosolic side but rather are transferred via the TOM complex into the intermembrane space and are subsequently directed to the outer membrane [32,33]. Therefore, protein N-myristoylation might positively affect these transport mechanisms of βbarrel proteins. Further studies are required to clarify the role of protein N-myristoylation on the targeting and integration of SAMM50 into the outer membrane of mitochondria. TOMM40L, an another outer membrane protein of mitochondria, is included in 18 proteins in which the product of full-length cDNA was found to be N-myristoylated in the insect cell-free protein synthesis system in this study. In addition, it was recently reported that TOMM40L is N-myristoylated in intact human cells [19,20]. Thus, it is possible that protein N-myristoylation positively affect the mitochondrial targeting of TOMM40L.
In the present study, in order to identify physiologically important human N-myristoylated proteins from cDNA resources, cell-free-and cellular metabolic labeling experiments coupled with bioinformatic prediction systems for protein N-myristoylation were performed using KOP human cDNA clones as a model cDNA clones. As a result, the products of 13 cDNA clones including many physiologically important proteins were found to be human N-myristoylated proteins. In this case, however, it should be noted that this method is useful to screen human N-myristoylated proteins, but the actual modification status and the role of the protein N-myristoylation on the protein function should be confirmed or studied in the intact human cells by the biochemical analyses such as those performed on SAMM50 in this study. Four proteins out of 13 N-myristoylated proteins found in this study are functionally unknown proteins. Since many of the N-myristoylated proteins play critical roles in cellular function, the finding of protein N-myristoylation may aid in the future functional characterization of these proteins.
The number of human proteins with an N-terminal Met-Gly sequence in all the human proteins listed in the Swiss-Prot protein knowledgebase (~46,000 proteins including isoforms) is approximately 3,700. Thus, it is expected that many physiologically important N-myristoylated proteins will be identified when the same approach is performed on larger cDNA resources that include many human cDNAs.