CHAPTER 12

Cellular Organelles and Membrane

Trafficking

© 2013 John Wiley & Sons, Inc. All rights reserved.

1
 Keys
• Emphasize the dynamic nature of the endomembrane system within the cell.
• Discriminate between regulated and constitutive secretion.
• Elucidate the structure and function of the rough and smooth ER.
• Outline events in synthesis and transport of membranes/proteins through the cell.
• Elucidate role and sites of glycosylation in processing of secretory/integral
membrane proteins.
• Elucidate the structure, function and polarization of the Golgi complex.
• Describe role of various types of coated- and non-coated-vesicles in membrane
trafficking.
• Describe the steps involved in the process of exocytosis.
• Describe lysosomal structure and function.
• Distinguish between phagocytosis, endocytosis and receptor-mediated
endocytosis.

© 2013 John Wiley & Sons, Inc. All rights reserved.

2
 Introduction
• Membranes divide the cytoplasm of eukaryotic cells
into distinct compartments.
• The endomembrane system includes organelles such
as:
– the endoplasmic reticulum,
– Golgi complex,
– endosomes,
– lysosomes, and
– vacuoles.

© 2013 John Wiley & Sons, Inc. All rights reserved.

3
(12.1) Overview of the Endomembrane System

Membrane-bound compartments of Inside vesicle: orientation

the cytoplasm remains the same

• 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.

© 2013 John Wiley & Sons, Inc. All rights reserved.

4
 Overview of the Endomembrane System
Biosynthetic and secretory pathways

• Several distinct pathways

through the cytoplasm have
been identified.
• Biosynthetic/ Secretory Endocytic
pathway – synthesis, pathways that
modification and transport unite
of proteins. endomembranes
• When proteins are into a dynamic,
discharged (secreted) from interconnected
network.
the cell.
– Constitutive secretion –
in a continuous fashion.
– Regulated secretion – in
response to a stimulus.

© 2013 John Wiley & Sons, Inc. All rights reserved.

5
Overview of the Endomembrane System
Synthesis and transport of secretory proteins
Autoradiography: reveals the sites of synthesis and subsequent
transport of secretory proteins.

3 minute pulse: radioactively 3 minute pulse: 3 minute pulse:

labeled amino acid (red) 17 minute chase 117 minute chase

• During regulated secretion, materials to be secreted are

stored in large, membrane-bound secretory granules.
– For example, secretion of digestive enzymes by pancreatic cells.
6
© 2013 John Wiley & Sons, Inc. All rights reserved.
 Study of Cytomembranes
GFP-based protein tracking
proteins can move
Use of Green Fluorescent protein stuck in ER
from ER to Golgi
Protein
– Green fluorescent
protein (GFP) – a
small protein isolated
from jellyfish which
emits green
fluorescent light.
– Fusing viral genes to
GFP gene allows the
study of protein traffic
due to large
production of
proteins.
The use of green fluorescent protein (GFP) reveals
the movement of proteins within a living cell.

© 2013 John Wiley & Sons, Inc. All rights reserved.

7
 (12.3) The Endoplasmic Reticulum

• The endoplasmic reticulum (ER) comprises a

network of membranes that penetrates much of the
cytoplasm.
• It is divided into two subcompartments:
– Rough endoplasmic reticulum (RER) & Smooth
endoplasmic reticulum (SER)
– In addition, the composition of the luminal or
cisternal space inside ER membranes is different
from the surrounding cytosolic space.

© 2013 John Wiley & Sons, Inc. All rights reserved.

8
(12.3) The Endoplasmic Reticulum
Rough ER

Smooth ER

© 2013 John Wiley & Sons, Inc. All rights reserved.

9
 The Endoplasmic Reticulum
• SER
– 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 from cytoplasm of
muscle cells.

© 2013 John Wiley & Sons, Inc. All rights reserved.

10
 The Endoplasmic Reticulum

Schematic diagram
showing stacks of
flattened cisternae
that make up the
rough ER

• RER:
– Composed of a network of flattened sacs (cisternae).
– Continuous with the outer membrane of the nuclear
envelope
– It has ribosomes on its cytosolic surface.
– Site of protein synthesis.

© 2013 John Wiley & Sons, Inc. All rights reserved.

11
 The Endoplasmic Reticulum
• Functions of the RER
– Synthesis of Proteins on Membrane-Bound versus Free
Ribosomes
• Approximately one-third polypeptides encoded by the
human genome are synthesized on ribosomes of RER
include: secreted proteins, integral membrane proteins,
and soluble proteins of organelles.
• Polypeptides synthesized on “free” ribosomes include:
cytosolic proteins, peripheral membrane proteins,
nuclear proteins, and proteins incorporated into
chloroplasts, mitochondria, and peroxisomes.

© 2013 John Wiley & Sons, Inc. All rights reserved.

12
 The Endoplasmic Reticulum
Synthesis of protein on membrane-bound ribosome

A schematic model of the synthesis of a secretory protein (or a

lysosomal enzyme) on a membrane-bound ribosome of the RER

• Synthesis of Secretory or Lysosomal protein on Membrane-Bound Ribosomes

– Messenger RNA binds to free ribosomes on cytosol.
– Secretory proteins synthesized on membrane-bound ribosomes have their signal
sequence recognized by a signal recognition particle (SRP)

© 2013 John Wiley & Sons, Inc. All rights reserved.

13
 The Endoplasmic Reticulum
Synthesis of protein on membrane-bound ribosome

• Binding to the ER occurs through two sequential interactions:

Initially, the SRP must interact with a SRP receptor.
Then the ribosome interacts with the translocon, which is a protein-lined
channel.
• Once the SRP-ribosome-nascent peptide chain complex binds ER, SRP is released.

© 2013 John Wiley & Sons, Inc. All rights reserved.

14
 The Endoplasmic Reticulum
Synthesis of protein on membrane-bound ribosome

• Processing of Newly Synthesized Proteins in the ER

– Upon entering the RER lumen, the signal sequence is cleaved by a signal
peptidase.
– Carbohydrates are added by the enzyme oligosaccharyltransferase.
– The RER lumen is packed with chaperones to assist in folding, and also
contains protein disulfide isomerase to add disulfide bonds to cysteines.

© 2013 John Wiley & Sons, Inc. All rights reserved.

15
 The Endoplasmic Reticulum

• Mechanisms that ensure the glucosyltransferase

destruction of misfolded
proteins
– 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 calreticulin
(ERAD), and ensures the
misfolded proteins do not
reach the cell surface.

Quality control: ensuring that misfolded

proteins do not proceed forward.

© 2013 John Wiley & Sons, Inc. All rights reserved.

16
 The Endoplasmic Reticulum

• Membrane Biosynthesis in the ER

– Membranes arise from pre-
existing membranes.
– Lipids are inserted into existing
membranes.
– As the membrane moves one
compartment to the next, its
proteins and lipids are modified.
– Membrane asymmetry is
established initially and
maintained during trafficking.

Maintenance of
membrane asymmetry

© 2013 John Wiley & Sons, Inc. All rights reserved.

17
 The Endoplasmic Reticulum
Modifying the lipid composition of membranes

• Contributing factors to
variation of organelle lipid
Schematic diagram
contribution showing three
– Organelle-specific distinct mechanisms:
enzymes for lipid 1. Enzymatic
conversion. modification
(head group)
– Inclusion/exclusion
2. Modification
process during vesicle during vesicle
formation. formation
– Lipid-transfer proteins 3. Modification by
that bind and transport phospholipid
lipids without the use of transfer proteins
vesicle transport.

© 2013 John Wiley & Sons, Inc. All rights reserved.

18
 The Endoplasmic Reticulum
Synthesis of the core portion of an oligosaccharide

Glycosylation in the RER

– Addition of sugars is
catalyzed by
glycosyltransferases.

Steps in the synthesis of the core portion of

an N-linked oligosaccharide in the rough ER

© 2013 John Wiley & Sons, Inc. All rights reserved.

19
 (12.4) The Golgi Complex

Schematic model of a portion of a EM: Golgi cisterna showing a

Golgi complex from an epithelial cell concave central domain and an
of the male rat reproductive tract. irregular peripheral domain

• The Golgi complex is a stack of flattened cisternae.

• It is divided into several functionally distinct compartments.
• The cis face of the Golgi faces the ER; the trans face is on the opposite
side of the stack.

© 2013 John Wiley & Sons, Inc. All rights reserved.

20
 The Golgi Complex
Regional differences in
membrane composition
across the Golgi stack:
1) Osmium tetroxide in
the cis cisternae
2) Mannosidase II in
the medial cisternae
3) Nucleotide
diphosphatase in
the trans cisternae

• The cis Golgi network (CGN) functions to sort proteins for the ER or the next Golgi
station.
• The trans Golgi network functions in sorting proteins either to the membrane or
various intracellular destinations.
• The Golgi complex is not uniform in composition; there are differences in
composition from the cis to the trans face.

© 2013 John Wiley & Sons, Inc. All rights reserved.

21
 The Golgi Complex

Glycosylation steps of a typical mammalian N-linked oligosaccharide in the Golgi

• Glycosylation in the Golgi Complex

– Assembly of carbohydrates found in glycolipids and glycoproteins takes place
in the Golgi.

© 2013 John Wiley & Sons, Inc. All rights reserved.

22
 The Golgi Complex

VSV
The dynamics
of transport
through the
Golgi complex
a-mann II

• The Movement of Materials through the Golgi Complex

– In the vesicular transport model, cargo is shuttled from the CGN to the TGN in
vesicles.
– In the cisternal maturation model, each cistern “matures” as it moves from the
cis face to the trans face.
– Current model: similar to cisternal maturation model but with vesicle retrograde
transport. Golgi cisternae serve an primary anterograde carriers.

© 2013 John Wiley & Sons, Inc. All rights reserved.

23
Types of Vesicle Transport and Their Functions
• Types of coated vesicles:
– COPII-coated vesicles – move materials from
the ER “forward” to the ERGIC and Golgi
complex.
– COPI-coated vesicles – move materials from
ERGIC and Golgi “backward” to ER, or from
the trans Golgi to the cis Golgi cisternae.
– Clathrin-coated vesicles – move materials
from the TGN to endosomes, lysosomes, and
plant vacuoles.

Proposed transport
between membrane
compartments of the
biosynthetic-
secretory pathway

© 2013 John Wiley & Sons, Inc. All rights reserved.

24
Types of Vesicle Transport and Their Functions

© 2013 John Wiley & Sons, Inc. All rights reserved.

25
Types of Vesicle Transport and Their Functions

• Sorting and Transport of

Lysosomal Enzymes Clatherin
– Lysosomal enzymes are coated
transported from the TGN in
clathrin-coated vesicles.

© 2013 John Wiley & Sons, Inc. All rights reserved.

26
 (12.6) Lysosomes

• Lysosomes contain acid hydrolases which

can digest every type of biological molecule.
• The low pH optimum of these enzymes is
maintained by a proton pump (H+-ATPase)

Portion of a phagocytic
Kupffer cell of the liver
showing at least 10
lysosomes of highly
variable size

© 2013 John Wiley & Sons, Inc. All rights reserved.

27
 Lysosomes
Autophagy
• Lysosomes play a key
role in organelle
turnover.
• During autophagy, an
organelle is surrounded
by a double membrane
and a structure called
an autophagosome is EM: a mitochondrion
produced. and peroxisome
• The autophagosome is enclosed in a double
membrane wrapper.
then fused with a
The autophagic vacuole
lysosome to produce (autophagosome) would
an autophagolysosome. have fused with a
• The digestive process A summary of the
lysosome and its
leaves a residual body. contents digested.
autophagic pathway

© 2013 John Wiley & Sons, Inc. All rights reserved.

28
 (12.7) Plant Cell Vacuoles

Cylindrical leaf cells Transmission

of the aquatic plant electron
Elodea with a large micrograph of
central vacuole a soybean
surrounded by a cortical cell
layer of cytoplasm showing the
containing the large central
chloroplasts vacuole

• A vacuole is a membrane-bound, fluid-filled compartment.

• Plant vacuoles have several storage functions.
• The vacuole membrane (tonoplast) contains an active transport system to
keep a high concentration of ions so that water enters by osmosis.
• Plant vacuoles contain acid hydrolases.

© 2013 John Wiley & Sons, Inc. All rights reserved.

29
Types of Vesicle Transport and Their Functions

• Exocytosis – discharge of a secretory vesicle or granule after fusion

with plasma membrane.
– Process is triggered by an increase in [Ca2+].
– Contacts between vesicle and plasma membrane lead to
formation “fusion pore”.
– The luminal part of the vesicle membrane becomes the outer
surface of the PM, and the cytosolic part becomes part of the
inner surface of the PM.

© 2013 John Wiley & Sons, Inc. All rights reserved.

30
 (12.8) The Endocytic Pathway: Moving
Membrane and Materials Into the Cell Interior

• Endocytosis – uptake of cell surface receptors and bound

extracellular ligands.
• Endocytosis can divided into :
– Phagocytosis – uptake of particulate matter.
– Pinocytosis – nonspecific uptake of extracellular fluids.
– Receptor-mediated endocytosis – uptake of specific
extracellular ligands following their binding to receptors.

© 2013 John Wiley & Sons, Inc. All rights reserved.

31
Copyright 2013 John Wiley & Sons, Inc.
All rights reserved. Reproduction or translation of this work
beyond that permitted in section 117 of the 1976 United
States Copyright Act without express permission of the
copyright owner is unlawful. Request for further information
should be addressed to the Permissions Department, John
Wiley & Sons, Inc. The purchaser may make back-up copies
for his/her own use only and not for distribution or resale.
The Publisher assumes no responsibility for errors,
omissions, or damages caused by the use of these programs
or from the use of the information herein.

© 2013 John Wiley & Sons, Inc. All rights reserved.

32