Endocytosis

The carrier and channel proteins discussed in the preceding section transport small
molecules through the phospholipid bilayer. Eukaryotic cells are also able to take up
macromolecules and particles from the surrounding medium by a distinct process
called endocytosis. In endocytosis, the material to be internalized is surrounded by an
area of plasma membrane, which then buds off inside the cell to form a vesicle
containing the ingested material. The term endocytosis was coined by Christian
deDuve in 1963 to include both the ingestion of large particles (such as bacteria) and
the uptake of fluids or macromolecules in small vesicles. The former of these
activities is known as phagocytosis (cell eating) and the latter as pinocytosis (cell
drinking).

Phagocytosis
During phagocytosis, cells engulf large particles such as bacteria, cell debris, or even
intact cells (Figure 12.34). Binding of the particle to receptors on the surface of the
phagocytic cell triggers the extension of pseudopodiaan actin-based movement of
the cell surface, discussed in Chapter 11. The pseudopodia eventually surround the
particle and their membranes fuse to form a large intracellular vesicle (>0.25 m in
diameter) called a phagosome. The phagosomes then fuse with lysosomes, producing
phagolysosomes in which the ingested material is digested by the action of lysosomal
acid hydrolases (see Chapter 9). During maturation of the phagolysosome, some of
the internalized membrane proteins are recycled to the plasma membrane, as
discussed in the next section for receptor-mediated endocytosis.
The ingestion of large particles by phagocytosis plays distinct roles in different kinds
of cells (Figure 12.35). Many amoebas use phagocytosis to capture food particles,
such as bacteria or other protozoans. In multicellular animals, the major roles of
phagocytosis are to provide a defense against invading microorganisms and to
eliminate aged or damaged cells from the body. In mammals, phagocytosis is the
function of primarily two types of white blood cells, macrophages and neutrophils,
which are frequently referred to as professional phagocytes. Both macrophages and
neutrophils play critical roles in the body's defense systems by eliminating
microorganisms from infected tissues. In addition, macrophages eliminate aged or
dead cells from tissues throughout the body. A striking example of the scope of this
activity is provided by the macrophages of the human spleen and liver, which are
responsible for the disposal of more than 1011 aged blood cells on a daily basis.

Receptor-Mediated Endocytosis
In contrast to phagocytosis, which plays only specialized roles, pinocytosis is
common among eukaryotic cells. The best-characterized form of this process is
receptor-mediated endocytosis, which provides a mechanism for the selective uptake
of specific macromolecules (Figure 12.36). The macromolecules to be internalized
first bind to specific cell surface receptors. These receptors are concentrated in
specialized regions of the plasma membrane, called clathrin-coated pits. These pits
bud from the membrane to form small clathrin-coated vesicles containing the
receptors and their bound macromolecules (ligands). The clathrin-coated vesicles then

fuse with early endosomes, in which their contents are sorted for transport to
lysosomes or recycling to the plasma membrane.
The uptake of cholesterol by mammalian cells has provided a key model for
understanding receptor-mediated endocytosis at the molecular level. Cholesterol is
transported through the bloodstream in the form of lipoprotein particles, the most
common of which is called low-density lipoprotein, or LDL (Figure 12.37). Studies
in the laboratories of Michael Brown and Joseph Goldstein demonstrated that the
uptake of LDL by mammalian cells requires the binding of LDL to a specific cell
surface receptor that is concentrated in clathrin-coated pits and internalized by
endocytosis. As discussed in the next section, the receptor is then recycled to the
plasma membrane while LDL is transported to lysosomes, where cholesterol is
released for use by the cell.
The key insights into this process came from studies of patients with the inherited
disease known as familial hypercholesterolemia. Patients with this disease have very
high levels of serum cholesterol and suffer heart attacks early in life. Brown and
Goldstein found that cells of these patients are unable to internalize LDL from
extracellular fluids, resulting in the accumulation of high levels of cholesterol in the
circulation. Further experiments demonstrated that cells of normal individuals possess
a receptor for LDL, which is concentrated in coated pits, and that familial
hypercholesterolemia results from inherited mutations in the LDL receptor. These
mutations are of two types. Cells from most patients with familial
hypercholesterolemia simply failed to bind LDL, demonstrating that a specific cell
surface receptor was required for LDL uptake. In addition, a few patients were
identified whose cells bound LDL but were unable to internalize it. The LDL
receptors of these patients failed to concentrate in coated pits, providing direct
evidence for the central role of coated pits in receptor-mediated endocytosis.
The mutations that prevent the LDL receptor from concentrating in coated pits lie
within the cytoplasmic tail of the receptor and can be as subtle as the change of a
single tyrosine to cysteine (Figure 12.38). Further studies have defined the
internalization signal of the LDL receptor as a sequence of six amino acids, including
the essential tyrosine. Similar internalization signals, frequently including tyrosine
residues, are found in the cytoplasmic tails of other receptors taken up via clathrincoated pits. These internalization signals bind to adaptor proteins, which in turn bind
clathrin on the cytosolic side of the membrane (Figure 12.39), similar to the way in
which clathrin-coated vesicles form during the transport of lysosomal hydrolases from
the trans Golgi network (see Figure 9.31). Clathrin assembles into a basketlike
structure that distorts the membrane, forming invaginated pits. A GTP-binding
protein, called dynamin, assembles into rings around the necks of these invaginated
pits, eventually leading to the release of coated vesicles inside the cell.
Receptor-mediated endocytosis is a major activity of the plasma membranes of
eukaryotic cells. More than 20 different receptors have been shown to be selectively
internalized by this pathway. Extracellular fluids are also incorporated into coated
vesicles as they bud from the plasma membrane, so receptor-mediated endocytosis
results in the nonselective uptake of extracellular fluids and their contents (fluid
phase endocytosis), in addition to the internalization of specific macromolecules.
Coated pits typically occupy 1 to 2% of the surface area of the plasma membrane and

are estimated to have a lifetime of 1 to 2 minutes. From these figures, one can
calculate that receptor-mediated endocytosis results in the internalization of an area of
cell surface equivalent to the entire plasma membrane approximately every 2 hours.
A variety of studies indicate that cells also possess clathrin-independent endocytosis
pathways. For example, fluids and some membrane-bound molecules continue to be
endocytosed under experimental conditions that inhibit endocytosis from clathrincoated pits. One pathway of clathrin-independent endocytosis involves the uptake of
molecules in small invaginations of the plasma membrane (50 to 80 nm in diameter)
called caveolae (Figure 12.40). Caveolae are enriched in lipid rafts of cholesterol and
sphingolipids and possess a distinct coat formed by a protein called caveolin. They
have been implicated in cell signaling and a variety of transport processes, including
endocytosis. In addition, large vesicles (0.15 to 5.0 m in diameter) can mediate the
uptake of fluids in a process known as macropinocytosis. Thus, while clathrindependent endocytosis clearly provides a major pathway for the uptake of both fluids
and specific macromolecules, cells also use several possible clathrin-independent
mechanisms.

Protein Trafficking in Endocytosis

Following their internalization, clathrin-coated vesicles rapidly shed their coats and
fuse with early endosomes, which are vesicles with tubular extensions located at the
periphery of the cell. The specificity of fusion of endocytic vesicles with endosomes
is determined by interactions between complementary pairs of transmembrane
proteins of the vesicle and target membranes (v-SNAREs and t-SNAREs) and by Rab
GTP-binding proteins, as discussed in Chapter 9. The early endosomes serve as a
sorting compartment, from which molecules taken up by endocytosis are either
recycled to the plasma membrane or transported to lysosomes for degradation. In
addition, the early endosomes of polarized cells can transfer endocytosed proteins
between different domains of the plasma membranefor example, between the apical
and basolateral domains of epithelial cells.
An important feature of early endosomes is that they maintain an acidic internal pH
(about 6.0 to 6.2) as the result of the action of a membrane H+ pump. This acidic pH
leads to the dissociation of many ligands from their receptors within the early
endosome compartment. Following this uncoupling, the receptors and their ligands
can be transported to different intracellular destinations. A classic example is provided
by LDL, which dissociates from its receptor within early endosomes (Figure 12.41).
The receptor is then returned to the plasma membrane via transport vesicles that bud
from the tubular extensions of endosomes. In contrast, LDL is transported (along with
other soluble contents of the endosome) to lysosomes, where its degradation releases
cholesterol.
Recycling to the plasma membrane is the major fate of membrane proteins taken up
by receptor-mediated endocytosis, with many receptors (like the LDL receptor) being
returned to the plasma membrane following dissociation of their bound ligands in
early endosomes. The recycling of these receptors results in the continuous
internalization of their ligands. Each LDL receptor, for example, makes a round-trip
from the plasma membrane to endosomes and back approximately every 10 minutes.
The importance of the recycling pathway is further emphasized by the magnitude of

membrane traffic resulting from endocytosis. As already noted, approximately 50% of

the plasma membrane is internalized by receptor-mediated endocytosis every hour
and must therefore be replaced at an equivalent rate. Most of this replacement is the
result of receptor recycling; only about 5% of the cell surface is newly synthesized per
hour.
Ligands and membrane proteins destined for degradation in lysosomes are transported
from early endosomes to late endosomes, which are located near the nucleus (see
Figure 12.41). Transport from early to late endosomes is mediated by the movement
of large endocytic carrier vesicles along microtubules. The late endosomes are more
acidic than early endosomes (pH about 5.5 to 6.0) and, as discussed in Chapter 9, are
able to fuse with transport vesicles carrying lysosomal hydrolases from the Golgi
apparatus. Late endosomes then mature into lysosomes as they acquire a full
complement of lysosomal enzymes and become still more acidic (pH about 5). Within
lysosomes, the endocytosed materials are degraded by the action of acid hydrolases.
Although many receptors (like the LDL receptor) are recycled to the plasma
membrane, others follow different fates. Some are transported to lysosomes and
degraded along with their ligands. For example, the cell surface receptors for several
growth factors (discussed in the next chapter) are internalized following growth factor
binding and eventually degraded in lysosomes. The effect of this process is to remove
the receptor-ligand complexes from the plasma membrane, thereby terminating the
response of the cell to growth factor stimulationa phenomenon known as receptor
down-regulation.
A specialized kind of recycling from endosomes plays an important role in the
transmission of nerve impulses across synapses (Figure 12.42). As discussed earlier in
this chapter, the arrival of an action potential at the terminus of most neurons signals
the fusion of synaptic vesicles with the plasma membrane, releasing the
neurotransmitters that carry the signal to postsynaptic cells. The empty synaptic
vesicles are then recovered from the plasma membrane in clathrin-coated vesicles,
which fuse with early endosomes. The synaptic vesicles are then regenerated directly
by budding from endosomes. They accumulate a new supply of neurotransmitter and
recycle to the plasma membrane, ready for the next cycle of synaptic transmission.
In polarized cells (e.g., epithelial cells), internalized receptors can also be transferred
across the cell to the opposite domain of the plasma membranea process called
transcytosis. For example, a receptor endocytosed from the basolateral domain of the
plasma membrane can be sorted in early endosomes for transport to the apical
membrane. In some cells, this is an important mechanism for sorting membrane
proteins (Figure 12.43). Rather than being sorted for delivery to the apical or
basolateral domains in the trans Golgi network (see Figure 9.28), proteins are initially
delivered to the basolateral membrane. Proteins targeted for the apical membrane are
then transferred to that site by transcytosis. In addition, transcytosis provides a
mechanism for the transfer of extracellular macromolecules across epithelial cell
sheets. For example, many kinds of epithelial cells transport antibodies from the blood
to a variety of secreted fluids, such as milk. The antibodies bind to receptors on the
basolateral surface and are then transcytosed along with their receptors to the apical
surface. The receptors are then cleaved, releasing the antibodies into extracellular
secretions.