The cell membrane is a flexible boundary made primarily of a lipid bilayer, which separates the cell’s interior from its environment. While many proteins span this membrane entirely to act as channels or transporters, another class of proteins interacts with the membrane surface without passing through it. A lipid-anchored protein is defined as a protein that is covalently attached to the membrane solely by a small lipid molecule inserted into the bilayer. Cells employ several distinct molecular methods to attach these proteins, each mechanism using a different lipid structure and targeting the protein to a specific side of the membrane.
The Purpose of Lipid Anchors
Lipid anchors provide unique functional advantages beyond simply having a protein span the membrane. Tethering a protein using only a lipid group allows it to remain highly mobile, moving laterally within the fluid membrane much faster than large, embedded proteins. This increased mobility is important for quickly clustering proteins together to initiate cell signaling events. Furthermore, the lipid anchor mechanism provides a regulated way for the cell to release the protein entirely. Specialized enzymes can cleave the lipid-protein bond, allowing the protein to be shed into the surrounding environment, which is utilized in communication and defense. Finally, the chemical nature of the anchor directs the protein to either the external side or the internal, cytosolic side of the membrane.
Attachment Through Glycosylphosphatidylinositol
One method for membrane attachment uses the Glycosylphosphatidylinositol (GPI) anchor, which tethers proteins exclusively to the exterior surface of the plasma membrane. The GPI anchor is a sophisticated glycolipid structure that functions as a spacer between the protein and the membrane’s outer leaflet. This anchor is composed of three main parts: a phosphatidylinositol lipid tail embedded in the membrane, a carbohydrate core, and a phosphoethanolamine linker that connects the core to the protein.
The process of adding the GPI anchor, known as glypiation, is a post-translational modification that takes place inside the endoplasmic reticulum (ER). An enzyme complex recognizes a specific signal sequence on the protein’s C-terminus, cleaves it off, and immediately replaces it with the pre-assembled GPI anchor. The newly anchored protein then travels through the secretory pathway to the cell surface. The anchor can be cleaved by specific phospholipases, allowing the protein to be released into the extracellular space, providing a mechanism for signal regulation and shedding surface components.
Attachment Through Fatty Acid Acylation
In contrast to the GPI anchor, fatty acid acylation methods attach proteins directly to the inner leaflet, the side facing the cell’s interior. These mechanisms involve the direct, covalent attachment of a single fatty acid chain to the protein, which significantly increases the protein’s hydrophobicity and affinity for the membrane. The two most common forms of this modification are myristoylation and palmitoylation.
Myristoylation is the stable, irreversible attachment of a 14-carbon fatty acid called myristate. This fatty acid is linked via an amide bond to a glycine residue that is the second amino acid from the protein’s N-terminus. The reaction occurs co-translationally, meaning it happens while the protein is still being synthesized. Because this bond is stable, the myristate group acts as a permanent tether to the inner membrane surface.
Palmitoylation is a dynamic and reversible process that links the 16-carbon fatty acid palmitate to a protein. This attachment forms a thioester bond with the sulfhydryl group of a cysteine residue. Because the thioester bond is chemically fragile, palmitate can be added by palmitoyltransferases and removed by thioesterases, allowing the protein to cycle on and off the membrane surface in response to cellular signals. Many proteins use dual acylation, where the stable N-terminal myristate modification is followed by a reversible palmitoylation on a nearby cysteine, ensuring the protein can be regulated but never fully loses its membrane affinity.
Attachment Through Prenylation
Another method for anchoring proteins to the inner leaflet involves prenylation, the attachment of isoprenoid lipids derived from the cholesterol synthesis pathway. This modification is chemically distinct from fatty acid acylation and is primarily used to anchor small G-proteins, like the Ras superfamily, which are important signaling molecules. Prenylation involves adding either a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group to a cysteine residue near the C-terminus of the protein.
The target site for prenylation is a sequence of four amino acids at the C-terminus known as the “CaaX box.” C is a cysteine, “aa” are two aliphatic amino acids, and X is the final amino acid. The identity of X determines which prenyl group is added; for example, if X is leucine, the protein is geranylgeranylated. The resulting isoprenoid lipid is attached via a stable thioether bond, which creates a highly hydrophobic tail that locks the protein to the cytosolic membrane surface. This modification is irreversible and is necessary for the proper function and membrane localization of proteins that regulate cell growth and division.