GO:0006499 N-terminal protein myristoylation: Protein Lipidation Pathway, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0006499 N-terminal protein myristoylation is the covalent attachment of a myristoyl group to the N-terminal amino acid residue of a protein, as defined by QuickGO.
• The reaction is catalyzed by N-myristoyltransferases (NMT1 and NMT2 in humans) and typically uses glycine as the N-terminal acceptor residue after removal of the initiator methionine.
• N-myristoylation is largely cotranslational in human cells and can also occur post-translationally after caspase cleavage or other proteolytic events.
• Quality control pathways, including a glycine-specific N-degron, recognize and degrade mis-myristoylated or unmyristoylated proteins to maintain proteostasis.
• N-myristoylation is essential for membrane targeting and signaling of many proteins and is a validated drug target in parasites such as Plasmodium falciparum.
• Modern research uses alkyne-tagged myristic acid, liquid-liquid extraction proteomics, and reconstituted cell-free systems to detect and quantify N-terminal myristoylation.
Description
N-terminal protein myristoylation (GO:0006499) is a lipid modification in which a 14-carbon saturated fatty acid, myristate, is covalently attached to the N-terminal amino acid of a protein. This modification is one of the most common N-terminal modifications in eukaryotes and is critical for protein-membrane interactions, subcellular targeting, and signal transduction. Because the myristoyl group is hydrophobic, its addition can anchor otherwise soluble proteins to lipid bilayers and regulate their function in processes ranging from immune signaling to cell death. Researchers study N-terminal myristoylation to understand how cells control protein localization and to develop therapeutics that interfere with this modification in pathogens and cancer. The reaction is catalyzed by N-myristoyltransferase (NMT) enzymes, which transfer myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins. In human cells, this event is predominantly cotranslational, meaning the myristoyl group is added while the protein is still being synthesized on the ribosome. However, post-translational myristoylation can occur after proteolytic exposure of an internal glycine, expanding the repertoire of modified proteins. Given its broad impact, N-terminal myristoylation is a focal point for proteomics, chemical biology, and CRISPR-based functional genomics.
N-terminal protein myristoylation At A Glance
| GO ID | GO:0006499 |
|---|---|
| GO term | N-terminal protein myristoylation |
| Ontology | biological_process |
| Synonym | none |
| Definition | The covalent attachment of a myristoyl group to the N-terminal amino acid residue of a protein. |
| Major function | Lipid modification that anchors proteins to membranes and regulates protein localization and signaling. |
| Enzymes involved | N-myristoyltransferases (NMT1, NMT2 in humans) |
| Substrate residue | Typically N-terminal glycine after initiator methionine removal |
| Myristate donor | Myristoyl-CoA |
| Subcellular context | Cotranslational on ribosomes and post-translational in the cytosol |
What Is GO:0006499?
According to the Gene Ontology, GO:0006499 N-terminal protein myristoylation is the biological process defined as the covalent attachment of a myristoyl group to the N-terminal amino acid residue of a protein. In practice, this means a myristate fatty acid is linked via an amide bond to the alpha-amino group of the first amino acid, which is usually glycine after the initiator methionine is removed. The modification is catalyzed by N-myristoyltransferase enzymes and uses myristoyl-CoA as the donor of the myristoyl group. This process is distinct from other lipid modifications such as palmitoylation or prenylation because it targets the N-terminus and uses a specific 14-carbon fatty acid.
Why Is N-terminal protein myristoylation Important in Cell Biology?
N-terminal protein myristoylation is important because it controls the membrane association and function of numerous key signaling proteins, and its dysregulation is linked to cancer, infectious disease, and developmental disorders. The modification is essential for the virulence of parasites such as Plasmodium falciparum, making N-myristoyltransferase an attractive drug target. In human cells, quality control pathways monitor myristoylation status, and defects can trigger protein degradation via a glycine-specific N-degron. Thus, understanding this process provides insights into basic cell biology and offers therapeutic opportunities.
• Controls membrane targeting of proteins involved in cell growth, apoptosis, and immune signaling.
• Essential for the intraerythrocytic development, egress, and invasion of Plasmodium falciparum, a malaria parasite.
• Misregulation of N-myristoylation is implicated in cancer progression and metastasis.
• A glycine-specific N-degron pathway mediates quality control of protein N-myristoylation, linking it to proteostasis.
• Provides a mechanism for reversible regulation of protein function through post-translational myristoylation after caspase cleavage.
• Serves as a target for chemical biology tools such as alkyne-tagged myristic acid for detection and proteomics.
• Enables cotranslational modification that can be studied in reconstituted cell-free systems.
• Offers a basis for translational medicine applications, including antiviral and anticancer strategies.
• Involved in the N-terminal modification landscape that affects protein stability and interactions.
• Facilitates proteomic profiling of myristoylated proteins using liquid-liquid extraction.
What Happens During N-terminal protein myristoylation?
Recognition of the N-terminal glycine motif
In simple terms: The enzyme looks for a specific tag at the start of a protein.
N-myristoyltransferase (NMT) recognizes substrate proteins that have a glycine residue at the N-terminus after the initiator methionine is removed. This glycine is part of a consensus motif that includes other small amino acids at positions 2, 5, and 6, which helps NMT bind the substrate. The recognition is highly specific, ensuring that only proteins with the correct N-terminal sequence are myristoylated.
Cotranslational myristoylation on the ribosome
In simple terms: The fat is attached while the protein is still being made.
In human cells, N-myristoylation occurs predominantly cotranslationally, meaning the myristoyl group is transferred to the growing polypeptide chain as it emerges from the ribosome. The enzyme NMT interacts with the ribosome and the nascent chain to catalyze the reaction efficiently. This coupling ensures that myristoylation happens before protein folding is complete, which can influence subsequent targeting and function.
Post-translational myristoylation after proteolytic cleavage
In simple terms: Sometimes the fat is added after a protein is cut.
Some proteins undergo post-translational myristoylation, where a proteolytic event such as caspase cleavage exposes a new N-terminal glycine that is then myristoylated. This allows for regulated addition of the myristoyl group in response to cellular signals, such as during apoptosis. This mode expands the range of myristoylated proteins beyond those with a canonical N-terminal glycine.
Quality control of myristoylation
In simple terms: Cells check if the fat was attached correctly and destroy mistakes.
A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. If a protein fails to be myristoylated or is incorrectly modified, it can be recognized by specific E3 ligases and targeted for degradation. This quality control ensures that only properly modified proteins reach their destinations.
Detection and quantification in research
In simple terms: Scientists use chemical tags and proteomics to find myristoylated proteins.
Researchers use alkyne-tagged myristic acid analogs to label and detect N-myristoylated proteins via click chemistry. Liquid-liquid extraction coupled with mass spectrometry enables proteome-wide profiling of N-terminal myristoylation. Reconstituted cell-free protein synthesis systems allow controlled study of regulated N-terminal modification.
Key Genes Involved in GO:0006499 N-terminal protein myristoylation
The following genes and proteins are central to N-terminal protein myristoylation, including the enzymes that catalyze the reaction and the substrates that are modified.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NMT1 | Catalyzes N-myristoylation of substrate proteins | Major enzyme for cotranslational myristoylation; target for inhibitor development |
| NMT2 | Catalyzes N-myristoylation of substrate proteins | Paralog of NMT1 with overlapping and distinct substrate specificity |
| GNAI1 | G protein alpha subunit, myristoylated substrate | Model substrate for studying membrane targeting and signaling |
| SRC | Non-receptor tyrosine kinase, myristoylated substrate | Involved in cancer signaling; myristoylation affects localization |
| c-Src | Cellular Src kinase, myristoylated substrate | Prototype for myristoylation-dependent membrane association |
| BID | BH3-interacting domain death agonist, post-translationally myristoylated | Apoptosis regulation; myristoylation after caspase cleavage |
| ARF1 | ADP-ribosylation factor 1, myristoylated substrate | Regulates vesicular trafficking; requires myristoylation for membrane binding |
| ARF6 | ADP-ribosylation factor 6, myristoylated substrate | Controls endocytic recycling and actin dynamics |
| NOS3 | Endothelial nitric oxide synthase, myristoylated substrate | Cardiovascular signaling; myristoylation targets to caveolae |
| FUS | RNA-binding protein, myristoylation reported | Neurodegeneration-related; N-terminal modifications may affect aggregation |
| HSP90AA1 | Chaperone, interacts with NMT | May assist in folding of myristoylated proteins |
| UBE2D1 | E2 ubiquitin-conjugating enzyme, involved in N-degron pathway | Quality control of myristoylation |
| UBR1 | E3 ubiquitin ligase, recognizes N-degron | Mediates degradation of mis-myristoylated proteins |
| UBR2 | E3 ubiquitin ligase, recognizes N-degron | Quality control of N-terminal modifications |
| UBR4 | E3 ubiquitin ligase, recognizes N-degron | Potential role in myristoylation quality control |
| Caspase-3 | Protease that generates post-translational myristoylation substrates | Apoptosis-induced myristoylation |
| Caspase-8 | Protease involved in apoptosis | May generate N-terminal glycine for myristoylation |
How Is N-terminal protein myristoylation Regulated?
N-terminal myristoylation is regulated at multiple levels. The expression and activity of N-myristoyltransferases (NMT1 and NMT2) can be modulated by cellular signals, although specific transcriptional regulators are not fully defined in the provided literature. Substrate availability and accessibility also influence the extent of modification, with cotranslational myristoylation depending on ribosome-nascent chain interactions. Post-translational myristoylation is regulated by proteolytic cleavage events, such as caspase activation during apoptosis, which expose new N-terminal glycines. Additionally, quality control pathways involving N-degrons and E3 ligases regulate the stability of myristoylated proteins, providing a layer of post-translational regulation.
N-terminal protein myristoylation and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NMT1 | Cancer; cell proliferation | Knockout or knockdown in cancer cell lines; xenograft models |
| NMT2 | Cancer; cell survival | Knockout in cancer cell lines; overexpression studies |
| SRC | Cancer; metastasis | Point mutation of N-terminal glycine to prevent myristoylation |
| BID | Apoptosis; cancer | Knock-in of myristoylation-deficient mutant; caspase cleavage studies |
| Plasmodium NMT | Malaria; parasite development | Parasite knockout or inhibitor treatment in erythrocyte cultures |
Cancer
N-myristoylation is critical for the function of several oncoproteins, including Src family kinases, which require myristoylation for membrane localization and signaling. Inhibition of N-myristoylation can reduce cancer cell proliferation and survival, making NMT a potential anticancer target. Dysregulation of myristoylated proteins has been observed in various cancers, although specific mutations in NMT genes are less common.
Infectious disease
Plasmodium falciparum, the malaria parasite, relies heavily on N-myristoylation for the function of proteins involved in host cell invasion and egress. Inhibition of protein N-myristoylation blocks intraerythrocytic development, egress, and invasion, highlighting NMT as a drug target for malaria. Other parasites may also depend on this modification, broadening the therapeutic potential.
Neurodegeneration
N-terminal modifications, including myristoylation, can influence protein aggregation and toxicity in neurodegenerative diseases. For example, myristoylation of certain proteins may affect their membrane interactions and contribute to neuronal dysfunction. However, direct evidence linking N-myristoylation to specific neurodegenerative disorders is still emerging.
From N-terminal protein myristoylation-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of NMT1 affect cell viability? | CRISPR knockout of NMT1 in human cell lines |
| Does preventing myristoylation of Src alter membrane localization? | Point mutation of N-terminal glycine to alanine in SRC |
| Can post-translational myristoylation of BID be tracked? | Knock-in of tagged BID with caspase cleavage site |
| What is the effect of NMT2 overexpression? | Overexpression of NMT2 in cell lines |
| How does myristoylation inhibition affect Plasmodium? | Parasite knockout of NMT or treatment with inhibitors |
| Can myristoylated proteins be enriched for proteomics? | Knock-in of alkyne-tagged myristic acid metabolic labeling |
How to Study the N-terminal protein myristoylation Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Alkyne-tagged myristic acid labeling | Incorporation of myristate analogs into proteins | Detection and enrichment of myristoylated proteins |
| Liquid-liquid extraction proteomics | Enrichment of N-terminal myristoylated peptides | Proteome-wide profiling of myristoylation |
| Cell-free protein synthesis | Cotranslational and post-translational myristoylation | Mechanistic studies of NMT activity |
| N-degron reporter assays | Degradation of mis-myristoylated proteins | Quality control pathway analysis |
| Click chemistry with fluorescent probes | Visualization of myristoylated proteins in cells | Imaging and subcellular localization |
| Mass spectrometry | Identification of myristoylation sites | Discovery of novel substrates |
| Western blot with anti-myristate antibodies | Presence of myristoylated proteins | Validation of specific candidates |
| In vitro NMT activity assay | Enzymatic transfer of myristate | Inhibitor screening and kinetics |
Detection with alkyne-tagged myristic acid
Alkyne-tagged myristic acid analogs can be metabolically incorporated into proteins and then detected via click chemistry with fluorescent or affinity tags. This method allows visualization and enrichment of N-myristoylated proteins from cell lysates. It is widely used for identifying new substrates and studying dynamics.
Liquid-liquid extraction proteomics
Liquid-liquid extraction coupled with mass spectrometry enables selective enrichment of N-terminal myristoylated peptides from complex mixtures. This approach provides a proteome-wide view of myristoylation sites and can quantify changes under different conditions. It is particularly useful for discovering novel substrates.
Cell-free protein synthesis
Reconstituted cell-free protein synthesis systems allow controlled addition of myristoyl-CoA and NMT to study N-terminal modification in vitro. This method can dissect the requirements for cotranslational versus post-translational myristoylation. It also enables the production of specifically modified proteins for functional studies.
Quality control assays
The glycine-specific N-degron pathway can be monitored using reporter proteins that are degraded when myristoylation is blocked. These assays help identify components of the quality control machinery and their regulation. They are valuable for studying proteostasis.
How CRISPR Can Be Used to Study GO:0006499 N-terminal protein myristoylation
Knockout
CRISPR knockout of NMT1 or NMT2 can reveal their essential roles in cell viability and protein myristoylation. Knockout of substrate genes, such as SRC, can be used to study the consequences of losing myristoylation on specific signaling pathways. These models are valuable for validating drug targets.
Point Mutation
Point mutation of the N-terminal glycine to alanine in substrate proteins prevents myristoylation and can be introduced via CRISPR to study localization and function. Such mutations help distinguish myristoylation-dependent from independent functions. They are also useful for creating separation-of-function alleles.
Knock-in
Knock-in of tagged or mutant versions of substrate proteins, such as BID with a caspase cleavage site, allows tracking of post-translational myristoylation. Knock-in of alkyne-tagged myristic acid metabolic enzymes can enhance labeling efficiency. These models provide precise tools for studying dynamics.
Overexpression
Overexpression of NMT1 or NMT2 can increase global myristoylation levels and may be used to study substrate saturation. Overexpression of myristoylated proteins can reveal gain-of-function phenotypes related to membrane targeting. These models are complementary to loss-of-function studies.
How EDITGENE Supports N-terminal protein myristoylation Research
Researchers studying N-terminal protein myristoylation-related genes often need to determine whether a candidate gene is causally involved in the modification, how mutations affect substrate recognition, and whether restoring or blocking myristoylation alters cellular phenotypes. EDITGENE provides a comprehensive suite of CRISPR-based services to address these questions with precision and scale.
Contact EDITGENE today to design your custom CRISPR model for N-terminal protein myristoylation research.
Frequently Asked Questions About N-terminal protein myristoylation
What is N-terminal protein myristoylation?
N-terminal protein myristoylation is the covalent attachment of a myristoyl group to the N-terminal amino acid residue of a protein, typically glycine, catalyzed by N-myristoyltransferases.
What genes are involved in N-terminal protein myristoylation?
Key genes include NMT1 and NMT2, which encode the enzymes, and substrate genes such as SRC, GNAI1, ARF1, and BID.
What is the GO ID for N-terminal protein myristoylation?
The Gene Ontology ID is GO:0006499.
How is N-terminal myristoylation detected?
It can be detected using alkyne-tagged myristic acid labeling, liquid-liquid extraction proteomics, or mass spectrometry.
Is N-terminal myristoylation cotranslational or post-translational?
In human cells it is predominantly cotranslational, but post-translational myristoylation occurs after proteolytic cleavage.
What diseases are linked to N-myristoylation?
It is linked to cancer, malaria, and potentially neurodegeneration, as it affects proteins involved in cell signaling and survival.
Can CRISPR be used to study N-myristoylation?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to study the enzymes and substrates of N-myristoylation.
What is the role of NMT1 and NMT2?
NMT1 and NMT2 are the enzymes that catalyze the transfer of myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins.
How does myristoylation affect protein localization?
Myristoylation anchors proteins to membranes, facilitating their localization to specific cellular compartments and enabling signaling.
What is the glycine-specific N-degron pathway?
It is a quality control pathway that recognizes and degrades proteins that fail to be properly myristoylated, maintaining proteostasis.
Conclusion
N-terminal protein myristoylation (GO:0006499) is a fundamental lipid modification that controls protein localization and function, with critical roles in health and disease. Understanding its mechanisms, substrates, and regulation offers opportunities for therapeutic intervention in cancer and infectious diseases. Advanced tools such as alkyne-tagged myristic acid and CRISPR-based models continue to drive discoveries in this field.
References
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- 3. Øye H et al.. 2025. Protein N-terminal modifications: molecular machineries and biological implications.. Trends Biochem Sci 50(4):290-310 PMID: 39837675
- 4. Tsumagari K et al.. 2023. Application of Liquid-Liquid Extraction for N-terminal Myristoylation Proteomics.. Mol Cell Proteomics 22(12):100677 PMID: 37949301
- 5. Xu Y et al.. 2023. Use of alkyne-tagged myristic acid to detect N-terminal myristoylation.. Methods Enzymol 684:191-208 PMID: 37230589
- 6. Matsumoto R et al.. 2023. Regulated N-Terminal Modification of Proteins Synthesized Using a Reconstituted Cell-Free Protein Synthesis System.. ACS Synth Biol 12(7):1935-1942 PMID: 37328154
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- 8. Schlott AC et al.. 2021. Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.. PLoS Biol 19(10):e3001408 PMID: 34695132
