Protein transport

Eukaryotic cells posses distinct membrane bound organelles which are absent in prokaryotic cells. The
membrane bound organelles have different functions and these organelles provide discrete compartments in
which specific cellular activities take place. The complex internal organization of eukaryotic cells generate
hardship for transport of proteins to their destinations.
Many proteins destined for the endoplasmic reticulum, the Golgi apparatus, lysosomes, the plasma
membrane and secretion from the cell are synthesized on ribosomes that are bound to the membrane of
endoplasmic reticulum (Figure-1).

Figure 1: Transport of proteins synthesized on membrane bound ribosomes
In contrast, proteins synthesized on free ribosomes either remain in the cytosol or are transported to the
nucleus, mitochondria, chloroplasts, or peroxisomes (Figure-2). The first step in the biosynthesis of secretory
proteins, plasma membrane proteins, and many other proteins in a eukaryotic cell involves the transport of at
least portions of the polypeptides across the endoplasmic reticulum membrane. Parts of the polypeptide chains
serve as signals that direct the translocation across and the integration into the endoplasmic reticulum
membrane and also determine the orientation of membrane proteins. Processing of the proteins takes place in
the endoplasmic reticulum and from the endoplasmic reticulum proteins are transported in vesicles to the Golgi
apparatus, where the proteins are further processed and sorted for transport to lysosomes, the plasma
membrane, or secretion from the cell.

Figure 2: Transport of proteins synthesized on free ribosomes
The Endoplasmic Reticulum
The endoplasmic reticulum (ER) is an organelle of cells in eukaryotic organisms that forms an interconnected
network of tubules, vesicles, and cisternae. It is the largest organelle of most eukaryotic cells. The general
structure of an endoplasmic reticulum is a membranous network of cisternae (sac-like structures) held together
by the cytoskeleton. The phospholipid membrane encloses a space, the cisternal space (or lumen), which is
continuous with the perinuclear space but separate from the cytosol. Two types of endoplasmic reticulum can
be distinguished i.e. rough endoplasmic reticulum and smooth endoplasmic reticulum (Figure-3).

Figure 3: Smooth and rough endoplasmic reticulum with nucleus
Rough endoplasmic reticula are involved in the synthesis of proteins and is also a membrane factory for
the cell, while smooth endoplasmic reticula are involved in the synthesis of lipids, including oils,
phospholipids and steroids, metabolism of carbohydrates, regulation of calcium concentration and
detoxification of drugs and poisons.

Protein targeting to the endoplasmic reticulum
In mammalian cells, most proteins are transferred into the endoplasmic reticulum while they are being
translated on membrane-bound ribosomes. In all eukaryotes, there are two pathways by which proteins can be
translocated into the endoplasmic reticulum i.e., a co-translational pathway and a post-translational
pathway. In the co-translational pathway, transport occurs while the polypeptide chain is being synthesized on
a membrane-bound ribosome, whereas in the post-translational pathway, the polypeptide chain is completed in
the cytoplasm before being transported into the endoplasmic reticulum. In prokaryotes, ribosomes don't seem
to be tightly bound to the membrane and most proteins may be transported post-translationally. Both
translocation modes require that polypeptides destined for translocation be specifically targeted to the
membrane.
In the co-translational pathway, first ribosomes are associated with the endoplasmic reticulum. Ribosomes
are targeted for binding to the endoplasmic reticulum by the amino acid sequence of the polypeptide chain
being synthesized rather than by the intrinsic properties of the ribosome itself. Proteins which are destined for
secretion are targeted to the endoplasmic reticulum by a signal sequence at the amino terminus of the growing
polypeptide chain. These signal sequences are short stretches of hydrophobic amino acids which are usually
cleaved from the polypeptide chain during its transfer into the lumen of the endoplasmic reticulum.
Mechanism: The signal sequences cover about twenty amino acids, including a stretch of hydrophobic
residues, usually at the amino terminus of the polypeptide chain. As soon as the signal sequences of the
growing polypeptide chain emerge from the ribosome, they are recognised and bound by a signal recognition
particle (SRP) consisting of six polypeptides and a small cytoplasmic RNA (srp RNA). Then the complex
containing the growing polypeptide chain, ribosome, and SRP is specifically targeted to the endoplasmic
reticulum membrane by an interaction with a membrane-bounrd receptor, the SRP receptor or docking protein
(Figure-4). In the next step, the SRP is released from both the ribosome and the signal sequence, where GTP
(guanosine triphosphate) plays a key role. The ribosome then binds to a protein translocation complex in the
membrane of the endoplasmic reticulum, and the signal sequence is inserted into a membrane channel or
translocon. The translocons are complexes of three transmembrane proteins, known as Sec61 proteins.
Transfer of the ribosome from the SRP to the translocon allows translation to resume, and the growing
polypeptide chain is transferred directly into the translocon channel and across the membrane of the
endoplasmic reticulum as translation proceeds. As translocation proceeds, the signal sequence is cleaved by the
signal peptidase and the polypeptide is released into the lumen of the endoplasmic reticulum. Finally, GTP
hydrolysis leads to the dissociation of the SRP from its receptor, and a new targeting cycle can begin. The
actual transfer of the polypeptide through the membrane does not require the SRP or its receptor and
commences only after their disengagement. Two basic functions are done by the SRP, where first it targets the
polypeptide chain to the Endoplasmic reticulum membrane by interacting both with the signal sequence and
with the translocation apparatus and secondly it keeps the bound signal sequence segregated from the rest of
the polypeptide chain and thereby prevents aberrant, premature folding.


Figure 4: The cotranslational pathway of transport of secretory proteins to the endoplasmic reticulum

Some proteins in mammals and many proteins in yeast are transported through post-translational pathway.
These proteins are synthesized on free cytosolic ribosomes and these proteins do not require a signal
recognition particle (SRP) for their transport. Their signal sequences are recognised by distinct receptor
proteins associated with the translocon in the endoplasmic reticulum membrane. The polypeptide chains are
remained in an unfolded conformation by the cytosolic Hsp70 chaperones.

Proteins inserted into the membrane of endoplasmic
reticulum
The proteins which are to be incorporated into the plasma membrane or the membrane of these compartments
are initially inserted into the membrane of endoplasmic reticulum instead of into its lumen. The proteins from
the membrane of endoplasmic reticulum travel the same pathway as secretory proteins. But along this pathway
these proteins are transported as membrane components.
Integral membrane proteins are embedded in the membrane by the hydrophobic regions that span the
phospholipid bilayer. The membrane spanning portions of the integral membrane proteins are usually -helical
regions consisting of 20-25 hydrophobic amino acids. Hydrogen bonding between the peptide bonds gets
maximized by the formation of an -helix and the hydrophobic amino acid side chains interact with the fatty
acid tails of the phospholipids in the bilayer. But there are different arrangement of different membrane
proteins such as some membrane proteins span the membrane only once whereas some span the membrane
more than once. Some proteins are oriented in the membrane with their amino terminus facing towards the
cytosol whereas some proteins are oriented with their carboxy terminus facing towards the cytosol. These
orientations of proteins inserted into the endoplasmic reticulum, Golgi bodies, lysosomes and plasma
membranes are established as the growing polypeptide chains are translocated into the endoplasmic reticulum.
After insertion into the endoplasmic reticulum membrane the transmembrane proteins are oriented with
their carboxy termini exposed to the cytosol. Before insertion of these transmembrane proteins into the
endoplasmic reticulum membrane, the normal amino-terminal signal sequence of these proteins, is cleaved by
signal peptidase during translocation of the polypeptide chain across the endoplasmic reticulum membrane
through the translocon. A second membrane-spanning -helix brings the polypeptide chain in the membrane.
This transmembrane sequence is called a stop-transfer sequence which signals closure of the translocon
channel. The carboxy-terminal portion of the growing polypeptide chain is synthesized in the cytosol as further
translocation of the polypeptide chain across the membrane of the endoplasmic reticulum is blocked.
Internal signal sequences that are not cleaved by signal peptidase also help in anchoring of proteins in the
endoplasmic reticulum membrane. These signal sequences act as transmembrane helices that exit the
translocon and anchor proteins in the membrane of the endoplasmic reticulum. Proteins inserted into the
membrane by the help of the internal signal sequences can have either their carboxy or amino terminus
exposed to the cytosol, because it depends upon the orientation of the signal sequences. Proteins that span the
membrane multiple times are thought to be inserted asa result of an alternating series of internal signal
sequences and transmembrane stop-transfer sequences.
Proteins are translocated across the endoplasmic reticulum membrane as unfolded polypeptide chains
during the progression of translation. These polypeptides fold into their three-dimensional conformation within
the endoplasmic reticulum by the help of molecular chaperones. Those proteins that cannot be correctly folded
are diverted from the secretory pathway and marked for degradation.

Transport of proteins from endoplasmic reticulum
Transport of proteins from endoplasmic reticulum to Golgi bodies occur through vesicles along the secretory
pathway. Vesicles are very small, membrane-enclosed sacs that carry cargo within the cell. Vesicles typically
transport large molecules that can not pass through a membrane on their own or by using other transport
molecules. Vesicles gather their cargo in a process called budding as they move with their cargo through the
cell and then deliver their cargo by fusing with another membrane enclosed compartment or with the cell's
plasma membrane. Vesicles bud-off from the endoplasmic reticulum and in the process capture the molecules
within the lumen of the endoplasmic reticulum. Proteins from the lumen of one organelle carried as budding
transport vesicle, released into the lumen of the recipient organelle following vesicle fusion. Vesicles that bud
from the transitional endoplasmic reticulum , carry their cargo first to the endoplasmic reticulum-Golgi
intermediate compartment and then to the Golgi apparatus (Figure-5). From Golgi apparatus, vesicular
transport occurs to lysosomes or the plasma membrane. The membrane proteins also undergo the same
pathway and their topological orientation is maintained as they travel from one membrane-bound organelle to
another.


Figure 5: Vesicular transport from the endoplasmic reticulum to the Golgi apparatus

In the transport pathway not all the proteins are transported but some proteins are retained within the
endoplasmic reticulum. Many proteins are retained in the endoplasmic reticulum lumen asa result of the
presence of the targeting sequence Lys-Asp-Glu-Leu (KDEL sequence) at their carboxy terminus. The
retention of some transmembane proteins in the endoplasmic reticulum is similarly detected by short C-
terminal sequences that contain two lysine residues (KKXX sequences). These signals cause resident
endoplasmic reticulum proteins to be selectively retrieved from the endoplasmic reticulum-Golgi intermediate
compartment or the Golgi complex and returned to the endoplasmic reticulum through a recycling pathway
(Figure-6). Proteins bearing these sequences bind to specific recycling receptors in the membranes of these
compartments and then selectively transported back to the endoplasmic reticulum.

The Golgi apparatus
The Golgi apparatus, also known as the Golgi complex or Golgi body, is an organelle found in most eukaryotic
cells. Part of the cellular endomembrane system, the Golgi apparatus packages proteins inside the cell before
they are sent to their destination; it is particularly important in the processing of proteins for secretion. The
Golgi apparatus is integral in modifying, sorting, and packaging these macromolecules for cell secretion
(exocytosis) or use within the cell. It primarily modifies proteins delivered from the rough endoplasmic
reticulum but is also involved in the transport of lipids around the cell, and the creation of lysosomes. The
Golgi plays an important role in the synthesis of proteoglycans, which are molecules present in the
extracellular matrix of animals. It is also a major site of carbohydrate synthesis. In plant cells, the Golgi
apparatus serves as the site at which the complex polysaccharides of the cell wall are synthesized.

Structure: The Golgi is composed of stacks of membrane-bound structures known as cisternae and associated
vesicles. Each cisterna comprises a flat, membrane enclosed disc that includes special Golgi enzymes which
modify or help to modify cargo proteins that travel through it. The cisternae stack has four functional regions:
the cis-Golgi network, medial-Golgi, endo-Golgi, and trans-Golgi network (Figure-6).


Figure 6: Structure of the Golgi apparatus showing vesicular transport

Vesicles from the endoplasmic reticulum are transported to the endoplasmic reticulum-Golgi intermediate
compartment and then enter the Golgi apparatus at the cis Golgi network. From the cis Golgi network, vesicles
progress to the medial and trans compartments of the Golgi stack, where most metabolic activities of the
Golgi apparatus take place. The modified proteins, lipids and polysaccharides then move to the trans Golgi
network, which acts as a sorting and distribution center directing to endosomes, lysosomes, the plasma
membrane and the cell exterior (Figure-6). Each region in the cisternae stack contains different enzymes which
selectively modify the contents depending on where they reside. The cisternae also carry structural proteins
important for their maintenance as flattened membranes which stack upon each other.

Protein processing within the Golgi
Processing of proteins begins with interaction of the newly made peptide with chaparone proteins in the lumen
of the endoplasmic reticulum. Chaparone proteins are released as the protein assumes its proper configuration.
For many secreted proteins (including cell surface proteins), the three dimensional structure of the protein is
supported by disulfide bridges that are formed between cysteine residues. Most proteins are modified in the
endoplasmic reticulum by addition of polysaccharides and the process is known as glycosylation. There are
two types of glycosylation i.e. N-linked glycosylation and O-linked glycosylation. The enzymes involved in
the glycosylation process are reside in the lumen of the endoplasmic reticulum. Initially in glycosylation, a pre-
fabricated oligosaccharide unit consisting of N-acetyl-glucosamine, mannose and glucose residues is
transferred from a glycolipid. The glycolipid consists of the oligosaccharide chain attached to dolichol
phosphate (a phospholipid with an extremely long hydrophobic chain), which serves as a membrane anchor for
the oligosaccharide chain. The oligosaccharide chain is attached to an asparagine residue through its free
amino group. The oligosaccharides are assembled on the cytosol side of the membrane and then flipped to the
lumen (cisterna) side of the membrane. The N-linked oligosachharides are generally undergo modification in
the Golgi apparatus. Subsequent to the addition of the 14 sugar oligosaccharide chain to the protein, there are a
long series of modifications of the polysaccharide chain, where Some of these modifiacations take place in the
ER, others in the cis Golgi network, other in the cis Golgi, others in medial Golgi and yet others in the trans
Golgi and trans Golgi network. Each of these steps is carried by a different enzyme that is localized in a
particular region of the Golgi. The processing of N-linked oligosaccharides is specific for the proteins destined
for specific regions, such as for plasma membrane or for secretion, and lysosomes.
N-linked oligosaccharides are processed in an ordered sequence of reactions within the Golgi apparatus.
The proteins which are destined for secretion or for the plasma membrane, are first modified by the removal of
three additional mannose residues which is followed by the sequential addition of an N-acetylglucosamine, the
removal of two more mannoses, and the addition of a fructose with two more N-acetylglucosamines. Finally
there is an addition of two galactoses and three sialic acid residues is made to it.
The processing of N-linked oligosaccharides of lysosomal proteins differs from that of plasma membrane
and secreted proteins. The lysosomal proteins are modified by mannose phosphorylation. First, there is
addition of N-acetylglucosamine phosphates to specific mannose residues, and this happens probably while the
protein is still in the cis Golgi network. After this N-acetylglucosamine group is removed, leaving mannose-6-
phosphate residues on the N-linked oligosaccharide. The phosphorylated mannose residues are specifically
recognised by a mannose-6-phosphate receptor in the trans Golgi network, which directs the transport of these
proteins to lysosomes.
Proteins can be modified by O-linked glycosylation, which involves the addition of carbohydrates to the
side chains of acceptor serine and thereonine residues within specific sequences of amino acids. These
modifications take place in the Golgi apparatus by the sequential addition of single sugar residues.

Transport of proteins from the Golgi apparatus
Proteins, lipids and polysaccharides are transported from the Golgi apparatus to their final destinations through
the secretory pathway. Proteins are sorted into different kinds of transport vesicles, which bud from the trans
Golgi network and deliver their contents to the appropriate cellular locations. Proteins that function within the
Golgi apparatus must be retained within that organelle, rather than being transported along the secretory
pathway. The proteins retained within the Golgi complex are associated with the Golgi membrane. The signals
responsible for retention of some proteins within the Golgi apparatus have been localized to their
transmembrane domains, which prevent the protein from being packaged in the transport vesicles that leave the
trans Golgi network. Some proteins are carried from the Golgi apparatus to the plasma membrane by a
constitutive secretory pathway and some proteins are transported to the cell surface by a distinct pathway of
regulated secretion or are specifically targeted to other intracellular destinations, such as lysosomes in animal
cells or vacuoles in yeast.
Proteins are sorted into the regulated secretory pathway in the trans Golgi network, where they are
packaged into specialized secretory vesicles. These immature secretory vesicles are larger than the transport
vesicles, often fuses with each other while further processing their protein contents. The sorting of proteins
into the regulated secretory pathway appears to involve the recognition of signal patches shared by multiple
proteins that enter this pathway.
In the process of selective transport of proteins to lysosomes, the lumenal lysosomal proteins are marked
by mannose-6-phosphates that are formed by modification of their N-linked oligosaccharides shortly after
entry into the Golgi apparatus. A specific receptor in the membrane of the trans Golgi network then recognizes
these mannose-6-phosphate residues. The resulting complexes of receptor with the lysosomal enzymes are
packaged into transport vesicles destined for lysosomes.
The cisternal maturation model proposed for transport of proteins through the Golgi apparatus deals with
the fact that the cisternae of the Golgi apparatus move by being built at the cis face and destroyed at the trans
face. Vesicles from the endoplasmic reticulum fuse with each other to form a cisterna at the cis face,
consequently this cisterna would appear to move through the Golgi stack when a new cisterna is formed at the
cis face. This model is supported by the fact that structures larger than the transport vesicles, such as collagen
rods, were observed microscopically to progress through the Golgi apparatus.
The vesicular transport model views the Golgi as a very stable organelle, divided into compartments in the
cis to trans direction. Membrane bound carriers transport material between the endoplasmic reticulum and the
different compartments of the Golgi. Experimental evidence includes the abundance of small vesicles also
known as shuttle vesicles in proximity to the Golgi apparatus. To direct the vesicles, actin filaments connect
packaging proteins to the membrane to ensure that they fuse with the correct compartment.
The cisternal maturation model and the vesicular transport model may actually work in conjuction with
each other and sometimes referred to as the combined model.

Vesicular transport
Transport vesicles play a central role in the traffic of molecules between different membrane-enclosed
compartments of the secondary pathway. The functional organization of the cell is maintained by the
selectivity of the vesicular transport.
The vesicles that leave the rough endoplasmic reticulum are transported to the cis face of the Golgi
apparatus, where they fuse with the Golgi membrane and empty their contents into the lumen. Once inside the
lumen, the molecules are modified, then sorted for transport to their next destinations. The Golgi apparatus
tends to be larger and more numerous in cells that synthesise and secrete large amounts of substances; for
example, the plasma B cells and the antibody-secreting cells of the immune system have prominent Golgi
complexes. Those proteins destined for areas of the cell other than either the endoplasmic reticulum or Golgi
apparatus are moved towards the trans face, to a complex network of membranes and associated vesicles
known as the trans-Golgi network (TGN). This area of the Golgi is the point at which proteins are sorted and
shipped to their intended destinations by their placement into one of at least three different types of vesicles,
depending upon the molecular marker they carry.
There are three different types of vesicles: exocytic vesicles(continous vesicles), secretory vesicles(
regulated vesicles) and lysosomal vesicles which take part in different types of secretion. Exocytic vesicles
contain proteins destined for extracellular release. The vesicles bud off after packaging and release their
contents into the extracellular space in a process known as constitutive secretion. Antibody release by
activated plasma B cells is an example of constitutive secretion. Secretory vesicles after packaging bud off
and are stored in the cell until a signal is given for their release. They move towards the membrane after
receiving the appropriate signal and fuse with the membrane to release their contents in a process also known
as regulated secretion. Release of neurotransmitters from neurons is an example of regulated secretion.
Lysosomal vesicles contain proteins destined for the lysosome, an organelle of degradation containing many
acid hydrolases, or to lysosome-like storage organelles. These proteins include both digestive enzymes and
membrane proteins. The vesicle first fuses with the late endosome, and the contents are then transferred to the
lysosome through an unknown mechanism.

Reconstituted vesicular transport
An understanding of the biochemical mechanisms that control the vesicular transport of proteins between
compartments of the secretory pathway of eukaryotic cells will require successful reconstitution of individual
segments of the pathway using novel cell-free systems. It is now possible to measure, in vitro, the transport of
proteins from the endoplasmic reticulum to the Golgi, between Golgi cisternae, and the formation of transport
vesicles en route from the trans Golgi network to the cell surface. The first cell-free transport system was
developed by James Rothman and collegues, who analyzed protein transport between compartments of the
Golgi apparatus. The experiment exploited a mutant mammalian cell line that lacked the enzyme required to
transfer N-acetylglucosamine residues to the N-linked oligosaccharide at an early stage of its modification in
the Golgi apparatus, as a result of which the glycoproteins produced by this mutant cell line lacked added N-
acetylglucosamine units. But when the Golgi stacks isolated from the mutant cell line were incubated with
stacks isolated from normal cells, N-acetylglucosamine residues were added to glycoproteins synthesized by
the mutant cells. The addition of N-acetylglucosamine provided a readily detectable marker for vesicular
transport in the reconstituted system. Similar reconstituted systems have been developed to analyze transport
between other compartments, including transport from endoplasmic reticulum to the Golgi, and transport from
the Golgi to secretory vesicles, vacuoles, and the plasma membrane.

The process of vesicle budding
There are different processes in the secretory pathway where transmembrane proteins and lumenal proteins and
their receptors are packaged into vesicles (Figure-7). The cargo proteins are first sorted from proteins targeted
for other destinations and the proteins that need to stay behind. A cargo- containing bud must develop on the
membrane and pinch off to form a transport vesicle and the transport vesicle must move to the target
membrane and fuse with it (Figure-7). The formation of most transport vesicles is regulated by small GTP-
binding proteins via adaptor proteins that directly interact with a vesicle coat protein. The sequential and
cooperative binding of GTP-binding proteins and adaptor proteins establishes a platform on the membrane for
a specific process, as a result of which assembly and budding of a transport vesicle directed from the trans
Golgi network to endosomes and lysosomes.


Figure 7: The process of vesicle budding and fusion

Most transport vesicles are coated with cytosolic proteins and termes as coated vesicles. The clatharin
coated vesicles are responsible for the uptake of extracellular molecules from the plasma membrane by
endocytosis as well as the transport of molecules from the trans Golgi network to endosomes and lysosomes.
The formation of clatharin-coated vesicles on the trans Golgi network requires clatharin, two types of adaptor
proteins, and a GTP-binding protein. Two other types of vesicles are also known as non clatharin-coated
vesicles (coat protein-coated vesicles) have been identified as budding from the endoplasmic reticulum and
Golgi complex. One class of these vesicles (COPII-coated vesicles) buds from the endoplasmic reticulum and
carries its cargo forward along the secretory pathway to the Golgi apparatus. Another class of these vesicles
(COPI-coated vesicles) bud from the endoplasmic reticulum-Golgi intermediate compartment and function in
the retrieval pathway that retain resident proteins in the Golgi and endoplasmic reticulum.

Vesicle fusion
For fusion of a transport vesicle with its target, the transport vesicle must recognise the correct target
membrane and the vesicle and target membranes must fuse and deliver the contents of the vesicle to the target
organelle (Figure-7). A hypothesis called SNARE hypothesis was proposed by James Rothman and
colleagues, in which vesicle fusion is mediated by interactions between specific pairs of transmembrane
proteins, called SNAREs, on the vesicle(v-SNAREs) and target membranes (t-SNAREs). According to this
hypothesis, the formation of complexes between v-SNAREs on the vesicle and t-SNAREs on the target
membranes lead to membrane fusion.
According to recent research, SNAREs are required for vesicle fusion with a target membrane and that
SNARE-SNARE pairing provides the energy to bring the two bilayers sufficiently close to destabilize them
and result in fusion.
Members of the Rab family of small GTP-binding proteins help in the docking of transport vesicles. They
function in several steps of vesicle trafficking, including interacting with SNAREs to regulate and facilitate the
formation of SNARE/SNARE complexes. To initiate transport vesicle fusion, Rab/GTP on the transport
vesicle recruits effector proteins and v-SNAREs to assemble a pre-fusion complex. A different Rab protein on
the target membrane similarly organizes other effector proteins and t-SNAREs. When the transport vesicle
encounters this target membrane, the effector proteins link the membranes by protein-protein interactions. This
tethering of the vesicle to the target membrane allows the v-SNAREs to contact the t-SNAREs. All SNARE
proteins have a long central coiled-coil domain and this domain binds strongly to other coiled coil domains and
so zips the SNAREs together, bringing the two membranes into nearly direct contact. This creates instability in
the lipid bilayers and as a result causing the fusion of the vesicles and the target membranes.

Endocytosis
Endocytosis is a process by which a cell engulfs some of its extracellular fluid including material dissolved or
suspended in it (Figure-8). Most substances that are important for cell are large polar molecules and so they
can not pass through the plasma membrane, because of which cells engulf them by endocytosis. In this
process the plasma membrane extends outward and surrounds the food particle. Endocytosis pathway can be
divided into major three categories:

Phagocytosis: If the material the cell takes in is particulate, such as a bacterium or a fragment of organic
matter, the process is called phagocytosis.
Pinocytosis: If the material the cell takes in is liquid, the process is called pinocytosis, otherwise known as cell
drinking process.

Receptor-mediated endocytosis: Specific molecules such as low-density lipoproteins are often transported
into eukaryotic cells through receptor mediated endocytosis. Molecules to be transported first bind to specific
receptors on the plasma membrane. The interior portion of the receptor protein is embedded in the membrane.
The protein clathrin coats the inside of the membrane in the area of the pit. When an appropriate collection of
molecules gather in the coated pit, the pit deepens and seals off to form a coated vesicle, which carries the
molecules into the cell.

Figure 8: The process of endocytosis

Components of an endocytic pathway
The endocytic pathway of mammalian cells consists of distinct membrane compartments that internalize
molecules from the plasma membrane and recycle them back to the surface or sort them to degradation.
Early endosomes: Early endosomes are often located in the periphery of the cell and receive most of types
of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular morphology and a mildly
acid pH. They are principally sorting organelles where many ligands dissociate from their receptors in the acid
pH of the lumen and from which many of the receptors recycle to the cell surface.
Late endosomes: Late endosomes often contain many membrane vesicles or membrane lamellae and
proteins characteristic of lysosomes, including lysosomal membrane glycoproteins and acid hydrolases. Late
endosomes are thought to mediate a final set of sorting events prior to delivery of material to lysosomes. Late
endosomes receive internalized material en route to lysosomes, usually from early endosomes in the endocytic
pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the
phagocytic pathway.
Lysosomes: Lysosomes are the last compartment of the endocytic pathway. These are membrane enclosed
organelles that contain an array of enzymes capable of breaking down all types of biological polymers i.e.,
proteins, nucleic acids, carbohydrates, and lipids. To do this task, These are then returned to the cytoplasm as
new cell-building materials. To accomplish the tasks, the lysosomes use different types of hydrolytic enzymes,
all of which are manufactured in the endoplasmic reticulum and modified in the Golgi apparatus. Lysosomes
function as the digestive system of the cell, serving both to degrade material taken up from outside the cell and
to digest obsolete components of the cell itself.
The formation of endosomes and lysosomes thus represents an interaction between the secretory pathway,
through which lysosomal proteins are processed, and the endocytic pathway through which extracellular
molecules are taken up at the cell surface. Material from outside the cell is taken up by clathrin-coated
endocytic vesicles, which bud from the plasma membrane and then fuse with early endosomes. Membrane
components are then recycled to the plasma membrane and the early endosomes gradually mature into late
endosomes, which are the precursors to lysosomes. Acid hydrolases are targeted to lysosomes by mannose-6-
phosphate residues, which are recognised by mannose-6-phosphate receptors in the trans Golgi network and
packaged into clathrin-coated vesicles. After the removal of the clathrin coat, these transport vesicles fuse with
late endosomes, and the acidic internal pH causes the hydrolases to dissociate from the mannose-6-phosphate
receptor. Then the hydrolases are released into the lumen of the endosome, while the receptors remain in the
membrane and are recycled to the Golgi. Then late endosomes are matured into lysosomes, which digest the
molecules taken up by endocytosis.