Chapter 12
Cellular Organelles and
Membrane Trafficking
1
•Endomembrane System
2
•Endoplasmic Reticulum
3
•Golgi Complex
4
•Lysosomes: Endocytosis
Overview
2
Includes
organelles such as
the endoplasmic
reticulum, Golgi
complex,
endosomes,
lysosomes, and
vacuoles
functioning as part
of a coordinated
unit.
Endomembrane system
3
• Organelles of the endomembrane system are
part of an integrated network in which
materials are shuttled back and forth.
• Materials are shuttled between organelles in
membrane-bound transport vesicles.
• Upon reaching their destination, the vesicles
fuse with the membrane of the acceptor
compartment.
Overview of the
Endomembrane System
4
• Biosynthetic pathway – synthesis,
modification and transport of proteins.
• Secretory pathway – when proteins are
discharged (secreted) from the cell.
• Constitutive secretion – in a continuous
fashion.
• Regulated secretion – in response to a
stimulus.
Distinct pathways
through the cytoplasm
5
During
regulated
secretion,
materials to be
secreted are
stored in large,
membranebound secretory
granules.
Endomembrane System
6
Autoradiography
Green Fluorescent Protein (GFP)
Biochemical analysis of subcellular fractions
Cell Free Systems
Mutant Study
Approaches to study
Cytomembranes
7
• A method to visualize biochemical
processes using radiolabeled materials
exposed to a photographic film.
• Can be used to determine where secretory
proteins are synthesized by using labeled
amino acids.
Autoradiography
8
• A small protein isolated from jellyfish which
emits green fluorescent light.
• A GFP-DNA chimera allows to observe the
protein synthesis in the cell.
• Viral genes fused to GFP allows the study of
protein traffic due to large production of
proteins
Use of Green
Fluorescent Protein
9
• Techniques to homogenize cells and isolate
some organelles, which can then be separated
from one another through subcellular
fractionation.
• Membrane vesicles derived from the
endomembrane system form a collection of
vesicles called microsomes, which can be
characterized further through other techniques.
Biochemical Analysis of
Subcellular Fractions
10
• Cell-free systems do not contain whole cells
and have provided information about the roles
of the proteins involved in membrane trafficking.
• Other type of information identified includes:
proteins that bind to the membrane to initiate
vesicle formation, and proteins responsible for
cargo selection.
Use of Cell-Free
Systems
11
• Mutants provide insights about the function of
normal gene products.
• Isolation of proteins from yeast has led to the
identification of homologous proteins in
mammals, pointing to the conserved nature of
endomembrane systems.
• Scientists can identify genes involved in a
particular process by determining which siRNAs
interfere with that process.
Study of Mutants
12
ENDOPLASMIC
RETICULUM
13
two components:
• Rough endoplasmic reticulum (RER)
• Smooth endoplasmic reticulum (SER)
• Different types of cells have different ratios of
the two types of ER, depending on activities
of the cell.
Endoplasmic reticulum
(ER)
14
• Composed of a network of flattened sacs
(cistenae).
• Continuous with the outer membrane of the
nuclear envelope and also has ribosomes on its
cytosolic surface.
RER
15
• Extensively developed in a number of
cell types.
• Functions include:
• Synthesis of steroid hormones in endocrine
cells.
• Detoxification in the liver of various organic
compounds.
• Sequestration of calcium ion into cytoplasm
of muscle cells.
SER
16
• Polypeptides synthesized on ribosomes of RER
include secreted proteins, integral membrane
proteins, and soluble proteins of organelles.
• Polypeptides synthesized on “free” ribosomes
include one-third of those encoded by the
human genome, cytosolic proteins, peripheral
membrane proteins, nuclear proteins, and
proteins incorporated into chloroplasts,
mitochondria, and peroxisomes.
Synthesis of Proteins on
Membrane-Bound versus Free
Ribosomes
17
• A signal sequence at N-terminus in secretory
proteins.
• Polypeptide moves into ER cisternal space
through a protein-lined pore.
• Movement through the membrane can occur
as it is being synthesized (cotranslationally) or
postranslationally.
Site of synthesis of a protein
determined by sequence of
amino acids in N-terminus.
18
Synthesis of protein on
membrane-bound ribosome
19
• Messenger RNA binds to free ribosomes on
cytosol.
• Secretory proteins synthesized on membranebound ribosomes have their signal sequence
recognized by a signal recognition particle (SRP)
• The SRP must interact with a SRP receptor.
• Another interaction must occur between the
ribosome and the translocon, which is a
protein-lined channel.
• Once the SRP-ribosome-nascent peptide chain
complex binds to the ER, the SRP is released
Synthesis of protein on
membrane-bound ribosome
20
• Upon entering the RER lumen, the signal
sequence is cleaved by a signal peptidase.
• Carbohydrates are added by the enzyme
oligosaccharltransferase.
• The RER lumen is packed with chaperones to
assist in folding, and also contains protein
disulfide isomerase to add disulfide bonds to
cysteines.
Processing of Newly
Synthesized Proteins in the ER
21
• Integral proteins contain hydrophobic transmembrane segments that interfere transfer into
the RER lumen.
• The translocon assists in the proper orientation
of transmembrane sequences.
• The arrangement within the membrane is
determined by the orientation of the first transmembrane segment is inserted.
Synthesis of Integral
Membrane Proteins
22
• Addition of sugars is catalyzed by glycosyltransferases.
• Core carbohydrate is modified by
oligosaccharyltransferase as the polypeptide is
transferred into the ER lumen.
• A glycoprotein goes through a system of quality
control to determine its fitness for a specific
compartment.
• Misfolded proteins are tagged by a terminal glucose
and recognized by chaperones for refolding.
Glycosylation in the
RER
23
• Misfolded proteins are not destroyed in the ER;
instead they are transported into the cytosol
where they are destroyed in proteasomes.
• This process is called ER-associated
degradation (ERAD), and ensures the misfolded
proteins do not reach the cell surface.
• Accumulation of misfolded proteins triggers the
unfolded protein response (UPR).
Destruction of
misfolded proteins
24