ENDOPLASMIC RETICULUM

Mammalian cells begin to import most proteins into the ER before complete synthesis of the
polypeptide chain—that is, import is a co-translational process. In contrast, the import of
proteins into mitochondria, chloroplasts, nuclei, and peroxisomes is a post-translational
process.

1) Smooth Endoplasmic Reticulum (SER) :

The surface of this type of reticulum is smooth as ribosome's not attached to it. Smooth ER is actively
engaged.

- Steroid synthesis like cholesterol, progesterone, testosterone, etc.

- Lipid synthesis.
- Glycogen synthesis.
- Metabolism of carbohydrates
- Detoxification function
- Major storage and released site of intercellular calcium ions

2) Rough Endoplasmic Reticulum (RER) : Rough Endoplasmic have ribosome attached throughout the
surface. These are present in cells, which are active in.

- protein synthesis.

-Protein translocation, folding, and transport of the protein.

- Glycosylation (this is the relation of a saccharide group with a hydroxyl or amino functional group to
form a glucoside).

-Disulfide bond formation (d-S-S bonds stabilize the tertiary and quaternary structures of many
proteins).

- Membrane synthesis.
MICROSOMES:

Translocation of secretory proteins across the ER membrane

- Free and membrane-bound ribosomes are functionally distinguishable and all proteins
synthesis initiate on the ribosome that is free in the cytosol. Ribosomes engaged with a
synthesis of a protein that is destined part of secretion are then entry to the ER by a signal
sequence at the N-terminal of the growing polypeptide chain.

- These signal sequence short stretches of 6-12 hydrophobic amino acids are usually cleaved
from the polypeptide chain into the ER lumen by N-signal peptidase.

- Some transport of most secretory protein into ER lumen occurs while the nascent protein is
still bound to the ribosome and being elongated, a process referred to as co-translational
modifications.

SIGNAL SEQUENCE HYPOTHESIS

The signal sequence hypothesis was first enunciated by Gunther Blobel who was awarded the Nobel
Prize in medicine in 1999 for his work.

As the nascent protein has emerged from the ribosome, a signal sequence is recognized and bound
by Signal Recognition Particle (SRP) a cytosolic ribonucleoprotein particle. Six discrete polypeptides
and 300 nucleotide RNA constitute the SRP.

SRP contains a small, cytoplasmic RNA. The SRP RNA binds with the ribosome as well as the targeting
signal sequence, inhibiting
further translation targeting the
entire complex translation Rough
ER by binding to the SRP receptor
on the ER membrane.

The ribosome binds to a protein

at the translocation complex, a
signal sequence is inserted into a
protein channel, sometimes
called translocons.
The entire process is coordinated by GTP binding to both SRP at SRP receptors.

For secretory proteins, the ER is the site of protein folding assembly of multisubunit protein, disulfide
bond formation, initial stages of glycosylation, and the addition of glycolipid anchors to some PM
proteins.

GOLGI COMPLEX

A Golgi apparatus is composed of flat sacs known as cisternae. The sacs are tacked in a bent
semicircular shape. Each stacked grouping has a membrane that separates its insides from the cell's
cytoplasm. Golgi membrane protein interactions are responsible for its unique shape. These
interactions generate the force that shapes this organelle. The Golgi apparatus is very polar. The
membrane at one end of the stack differs in composition and in thickness from those at the other
end. One end (Cis face) acts as the "receiving" department while the other (trans face) acts as the
"shipping" department. The Cis face is closely associated with the ER.

The Golgi apparatus is made up of a series of a compartment of single membrane-bound organelle

and part of the endomembrane system. It consists of two main networks i) Cis Golgi network (GCN),
II) trans Golgi network (TGN). The GCN is a collection of fused, flattened membrane-enclosed disks
known as cisternae, originating from vernacular clusters that bud off from the endoplasmic reticulum

It is a major site of carbohydrate synthesis as well as the final sorting and dispatching center for
products of the endoplasmic reticulum. While in the endoplasmic reticulum, many proteins undergo
the first stage of glycosylation. Most proteins then migrate inside the vesicle from ER and enter the
cis-face of the Golgi. Golgi membrane is studied with glycosyltransferase, glycosidases, and
nucleotide sugar transporter arranged in a generally ordered manner from the cis-Golgi to the trans-
Golgi network. (TGN), such that each activity is able to act on a specific substrate generated earlier in
the pathway.

High mannose oligosaccharide is trimmed but has no new sugars added to them in the Golgi
apparatus.

Vesicle transport and signal sequences –

The most important vesicles that carry secretory protein from ER to Golgi and from Golgi to other
targets are coated with a cytosolic coat protein and thus are called coated vesicles.
The budding of vesicles from ER to GOLGI is assisted by COP-II. After budding out of ER, the vesicles
lose their COP II coat and fuse together to form the tubular clusters. This fusion is promoted by the v-
SNARE & t-SNARE of the vesicles.

While vesicles moving from GOLGI to ER (retrieval transport) utilized COP-I. Retrieval signals are
usually located at the C-terminal end of the protein.

For ER membrane proteins, it is usually KKXX (Lys-Lys-X-X)-This sequence is present on KDEL

receptors (multipass transmembrane protein) to bind the COPI coat.

For soluble ER-resident proteins, it is usually KDEL (Lys-Asp-Glu-Leu or something similar). KDEL
receptors have a high affinity for KDEL tubular clusters and Golgi, while the KDEL receptor has a low
affinity for KDEL in ER (including its release in ER).

There is a pH gradient from ER (neutral) to Golgi (acidic).

This pH difference is regulated by V-type ATPases (proton pumps).

The pH effect plays a crucial role in KDEL receptor binding: 1. Low pH, high affinity 2. Neutral pH, low
affinity

But not all proteins have retrieval signals.

The third type of COP protein clathrin is required for the budding of vesicles from PM and possibly
from the trans-Golgi. Clathrin-coated vesicles are best understood and are responsible for uptake of
extracellular molecules from PM by endocytosis as well as transport of molecules from TGN to the
endosome, lysosome, or to the PM. Thus, clathrin-coated vesicles participate in endocytic recycling,
retrograde transport the bring soluble and membrane-bound proteins into the cell. Before a vesicle
fuse, with a target membrane, discard its coat.
Clathrin-coated vesicles, the first coated vesicles to be identified, transport mate - rial from the
plasma membrane and between endosomal and Golgi compart - ments. COPI-coated vesicles and
COPII-coated vesicles transport material early in the secretory pathway:

COPI-coated vesicles bud from Golgi compartments, and COPII-coated vesicles bud from the ER

Rab Proteins Guide Transport Vesicles to Their Target Membrane

Rab proteins play a central part in the specificity of vesicle transport. Rab proteins are also
monomeric GTPases. With over 60 known members, the Rab subfamily is the largest of the
monomeric GTPase subfamilies

The SNARE proteins (also called SNAREs, for short) catalyze the membrane fusion reactions in vesicle
transport. There are at least 35 different SNAREs in an animal cell, each associated with a particular
organelle in the secretory or endocytic pathways. These transmembrane proteins exist as
complementary sets, with v-SNAREs usually found on vesicle membranes and t-SNAREs usually found
on target membranes

ENDOCYTOSIS:

Eukaryotic cells are continually taking up fluid, along with large and small molecules, by the
process of endocytosis.

The material to be ingested is progressively enclosed by a small portion of the plasma

membrane, which first buds inward and then pinches off to form an intracellular endocytic
vesicle. The ingested materials, including the membrane components, are delivered to
endosomes, from which they can be recycled to the plasma membrane or sent to lysosomes for
digestion.

Two main types of endocytosis are distinguished on the basis of the size of the endocytic vesicles
formed.

1. Pinocytosis (“cellular drinking”) involves the ingestion of fluid and molecules via small
pinocytic vesicles (250 nm in diameter).

2. Large particles are ingested mainly by specialized phagocytic cells and the process is
phagocytosis. In protozoa, phagocytosis is a form of feeding: these unicellular eukaryotes ingest
large particles such as bacteria by taking them up into phagosome. Phagocytic cells—including
macrophages, which are widely distributed in tissues, and other white blood cells, such as
neutrophils—defend us against infection by ingesting invading microorganisms.

Receptor mediated Endocytosis

Cholesterol is a lipid that is extremely insoluble in water. It is transported in the bloodstream

bound to proteins in the form of particles called low-density lipoproteins, or LDL. Cholesterol-
containing LDLs, which are secreted by the liver, bind to receptors located on the surface of cells.
The resulting receptor–LDL complexes can then be ingested by receptor-mediated endocytosis
and delivered to endosomes. The interior of endosomes is more acidic than the surrounding
cytosol or the extracellular fluid, and in this acidic environment the LDL dissociates from its
receptor: the empty receptors are returned, via transport vesicles, to the plasma membrane for
reuse, while the LDL is delivered to lysosomes. In the lysosomes, the LDL is broken down by
hydrolytic enzymes. Freed from the bulky LDLs, cholesterol escapes into the cytosol, where it
can be used to synthesize new membrane.
LYSOSOMES:

Lysosomes are membranous sacs of hydrolytic

enzymes that carry out the controlled intracellular
digestion of both extracellular materials and worn-
out organelles. They contain about 40 types of
hydrolytic enzymes, including those that degrade
proteins, nucleic acids, oligosaccharides, and
lipids. All of these enzymes are optimally active in
the acidic conditions (pH ~5) maintained within
lysosomes. The membrane of the lysosome
normally keeps these destructive enzymes out of
the cytosol (whose pH is about 7.2), but the
enzymes’ acid dependence protects the contents
of the cytosol against damage should some of
them escape.

The lysosomal membrane contains transporters

that allow the final products of the digestion of
macromolecules, such as amino acids, sugars, and
nucleotides, to be transferred to the cytosol; from
there, these materials can be either exported or
utilized by the cell. The membrane also contains
an ATP-driven H+ pump, which, like the ATPase in
the endosome membrane, pumps H+ into the lysosome, thereby maintaining its contents at an
acidic pH (Figure 15−36).

Most of the lysosomal membrane proteins are unusually highly glycosylated; the sugars, which
cover much of the protein surfaces facing the lysosome lumen, protect the proteins from
digestion by the lysosomal proteases.

The specialized digestive enzymes and membrane proteins of the lysosome are synthesized in
the ER and transported through the Golgi apparatus to the trans Golgi network. While in the ER
and the cis Golgi network, the enzymes are tagged with a specific phosphorylated sugar group
(mannose 6-phosphate), so that when they arrive in the trans Golgi network they can be
recognized by an appropriate receptor, the mannose 6-phosphate receptor. This tagging
permits the lysosomal enzymes to be sorted and packaged into transport vesicles, which bud off
and deliver their contents to lysosomes via endosomes.

Cells have an additional pathway that supplies materials to lysosomes; this pathway, called
autophagy, is used to degrade obsolete parts of the cell: as the term suggests, the cell literally
eats itself.
 The Mannose-6-Phosphate Receptor

The mannose-6-phosphate receptor sorts proteins into clathrin-coated vesicles that are leaving the
trans-Golgi network and are destined for organelles called lysosomes involved in breaking down
cellular waste products.

Mannose-6-phosphate is an N-linked oligosaccharide generated by enzymes present in the cis-Golgi

cisterna. It is formed specifically on proteins destined for the lysosome.

The mannose-6-phosphate receptor spans the Golgi membrane and binds to coat proteins on the
cytosolic side of the membrane and to the mannose-6-phosphate-containing protein on the luminal
side of the membrane. Thus, mannose-6-phosphate-containing proteins are sorted and concentrated
into the correct clathrin-coated vesicles for transport to the lysosome.