BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February

17, 2023.
Intracellular Compartments
Membrane Enclosed Organelles
Eukaryotic cells contain a basic set of membrane enclosed organelles These compartments can
separate metabolic processes The compartments can be observed in this electron micrograph
of a liver cell.

In eukaryotic cells, internal

membranes create enclosed
compartments that segregate different
metabolic processes.
Examples of many of the major
membrane-enclosed organelles can be
identified in this electron micrograph of
part of a liver cell, seen in cross section.
The small, black granules between the
compartments are aggregates of
glycogen and the enzymes that control
its synthesis and breakdown.

Organelles have distinctive forms. Each is separated from the cytoplasm by at least one
phospholipid bilayer A distinct region of the cell, such as an organelle, or even a distinct region
of the membrane, can be considered a compartment
cell from the lining of the intestine
contains the basic set of membrane-
enclosed organelles found in most
animal cells.
The nucleus, endoplasmic reticulum (ER),
Golgi apparatus, lysosomes, endosomes,
mitochondria, and peroxisomes are
distinct compartments separated from
the cytosol by at least one selectively
permeable membrane. Ribosomes are
shown bound to the cytosolic surface of
portions of the ER, called the rough ER;
the ER that lacks ribosomes is called
smooth ER. Additional ribosomes can be
found free in the cytosol.
- Video
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.

The numbers of
some organelles vary
depending on cell
type.

The membrane of each organelle encloses a specific set of large and small molecules,
including many proteins, that carry out a variety of functions.
How do these molecules come to reside in the organelle?
Ions and small molecules use channel and carrier proteins What about proteins…
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Protein sorting

Proteins move around within the cell using several different mechanisms. They are transported
into organelles in three distinct ways:
1. Transport through nuclear pores
2. Transport across membranes
3. Transport by vesicles
Membrane-enclosed organelles import
proteins by one of three mechanisms.
All of these processes require energy. The
protein remains folded during transport in
mechanisms 1 and 3 but usually has to be
unfolded during mechanism 2.
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Proteins are targeted to specific organelles by amino acid sequences that act like “postal
codes”. These are called signal peptides or signal patches Gunter Blobel won the Nobel Prize in
1999 "for the discovery that proteins have intrinsic signals that govern their transport and
localization in the cell
- There are specific amino acids at the N terminus (signal peptide) and they only at at
specific times

- Video
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
There are distinct signal sequences for each organelle

The importance of signal sequences can be studied by engineering proteins with an attached
sequence and identifying where they locate
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
How do proteins get into and out of the nucleus
- Transport through nuclear pores (section 1 below)
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Sorting to the nucleus

The nucleus is surrounded by two membranes, together

forming the nuclear envelope
The outer membrane is continuous with the ER
The envelope is perforated by nuclear pores, which control
what enters and exits The outer nuclear membrane is
continuous with the ER membrane.
The double membrane of the nuclear envelope is
penetrated by nuclear pores. The ribosomes that are
normally bound to the cytosolic surface of the ER membrane
and outer nuclear membrane are not shown

A nuclear pore is a complex of about 30 proteins, that act like a gate and are arranged in such a
way as to allow small molecules through but selectively control large ones

An illustration shows a section of

nuclear envelope with the cytosol, a
part of nucleus, and pores on a scale
of 50 nanometers. The labeled parts
are nuclear basket, nuclear pore
complex protein, nuclear lamina,
inner nuclear membrane, outer
nuclear membrane, and cytosolic
fibrils.
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Electron micrographic views of nuclear
pores from the side (top) and face-on
(bottom figure)

The nuclear pore complex forms a gate

through which selected
macromolecules and larger complexes
enter or exit the nucleus.

RNA and ribosomal subunits must exit the

nucleus and proteins formed in the cytoplasm
need to enter
The latter are directed to the nucleus by a
nuclear localization signal
Nuclear import receptors bind to the signal
sequence and guide the protein through the pore
Prospective nuclear proteins are imported from
the cytosol through nuclear pores.
The proteins contain a nuclear localization signal
that is recognized by nuclear import receptors,
which interact with the cytosolic fibrils that
extend from the rim of the pore. After being
captured, the receptors with their cargo jostle
their way through the gel-like meshwork formed
from the unstructured regions of the nuclear pore
proteins until nuclear entry triggers cargo release.
After cargo delivery, the receptors return to the
cytosol via nuclear pores for reuse. Similar types
of transport receptors, operating in the reverse
direction, export mRNAs from the nucleus (see
Figure 7–25). These sets of import and export
- Video
receptors have a similar basic structure.
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Figure 15–10b Energy supplied by GTP hydrolysis drives nuclear transport.
A nuclear import receptor picks up a prospective nuclear protein in the cytosol and enters the
nucleus. There it encounters Ran-GTP, which binds to the import receptor, causing it to release
the nuclear protein. Having discharged its cargo in the nucleus, the receptor—still carrying Ran-
GTP—is transported back through the pore to the cytosol, where Ran hydrolyzes its bound GTP.
Ran-GDP falls off the import receptor, which is then free to bind another protein destined for
the nucleus. Ran-GDP is carried into the nucleus by its own unique import receptor (not
shown).

The energy required for movement through the pores is derived from GTP hydrolysis, aided by
a GTPase called Ran.
Nuclear import receptor binds Ran-GTP When the GTP is hydrolyzed, it falls off and a protein
with a nuclear localization signal can bind This complex can then move through the nuclear
pore Inside the nucleus, Ran-GTP binds to the receptor, and the nuclear protein is delivered to
the nucleus.
 Nuclear import receptor binds Ran-GTP
 When the GTP is hydrolyzed, it falls off and a protein with a nuclear localization signal
can bind
 This complex can then move through the nuclear pore Inside the nucleus,
 Ran-GTP binds to the receptor, and the nuclear protein is delivered to the nucleus.
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.

GTPases make up a family of proteins that bind the

nucleotide guanosine triphosphate (GTP) and
hydrolyze it to GDP and inorganic phosphate
Here, Ran cycles between GDP and GTP bound forms,
with the aid of other enzymes (Ran-GAP and RanGEF)
Similar cyclical reactions drive many processes in the
Cell
An Illustration shows nuclear transport driven by
G TP hydrolysis. Ran-G T P is inside the nucleus and
Ran-G D P is inside the cytosol. Ran-G E F (guanine
nucleotide exchange factor) in the nucleus causes the release of G D P of Ran-G D P and the
addition of G T P inside the nucleus. Ran-G A P (G T P ase-activating protein) in the cytosol
converts Ran-G T P to Ran-G D P, with the release of a phosphate.
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
Protein sorting to the mitochondria
How do proteins move across membranes into the mitochondria,
chloroplasts, peroxisomes and endoplasmic reticulum?
Mitochondria and ER will be used as examples.

Mitochondria are also enclosed by a double membrane

Most mitochondrial proteins are synthesized in the cytosol from nuclear genes and
translocated to the mitochondrial matrix
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
1. Mitochondrial proteins are synthesized in the cytosol
2. Protein with a signal sequence binds to an import receptor protein in the outer
membrane

Signal peptide is cleaved

3. Protein moves into intermembrane space with the aid of a translocator in the outer
membrane
4. The complex diffuses through the membrane and meets a translocator in the inner
membrane
5. Protein moves into mitochondrial matric and signal peptide is cleaved
6. Also required chaperone proteins and ATP (not shown)
- Video

Sorting to ER
The endoplasmic reticulum (ER) is an extensive organelle that’s involved in the synthesis of
proteins destined for many other locations

The ER is often studded with

ribosomes that are
synthesizing proteins. There
is also a smooth ER devoid of
ribosomes.

ER Tubules – video
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
A common pool of ribosomes is used to synthesize all proteins encoded by the nuclear genome.
mRNA that has exited the nucleus will begin to be translated on free ribosomes
Ribosomes that are translating proteins with an ER signal sequence (red) are directed to the ER
membrane, otherwise they remain in the cytosol. Many ribosomes translate each mRNA
(polyribosomes)
Goes across 5’ to 3’
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
- A signal recognition particle (SRP) binds to the signal sequence and ribosome, pausing
translation and then interacts with an SRP receptor in the ER membrane.
- The SRP is released and the ribosome passes from the SRP receptor to a protein
translocator (light blue) in the ER membrane
- Protein synthesis resumes, and the translocator starts to transfer the growing
polypeptide across the lipid bilayer

An ER signal sequence and an SRP direct a ribosome to the ER membrane.

The SRP (brown) binds to both the exposed ER signal sequence and the ribosome, thereby
slowing protein synthesis by the ribosome. The SRP–ribosome complex then binds to an SRP
receptor (dark blue) in the ER membrane. The SRP is released, and the ribosome passes from
the SRP receptor to a protein translocator (light blue) in the ER membrane. Protein synthesis
resumes, and the translocator starts to transfer the growing polypeptide across the lipid bilayer

Proteins enter the ER while they are being synthesized by the ribosome (not shown)
Soluble proteins cross the ER membrane and are released into the lumen
Signal peptide is cleaved off by a signal peptidase (yellow), remains in the membrane and gets
degraded
BIOL 2070: Cell Biology Lecture 6: Intracellular Compartments and Protein Transport February
17, 2023.
In addition to the ER signal sequence, stop transfer sequences (shown in orange) can stop the
translocation of proteins, resulting in a transmembrane protein in the lipid bilayer
After the stop transfer sequence enters the bilayer, the rest of the protein is translated in the
cytosol

- The orange part stops transfer now there is a protein on either side of the membrane.
A double-pass transmembrane protein will have an internal ER signal sequence that acts as a
start-transfer sequence
The start and stop sequences don’t get cleaved off and remain part of the protein
Summary