Alberts • Bray • Hopkin • Johnson • Lewis • Raff • Roberts • Walter

Essential
Cell Biology
FOURTH EDITION

Chapter 15
Intracellular Compartments and
Protein Transport
Copyright © Garland Science 2014
MEMBRANE-ENCLOSED ORGANELLES
Eukaryotic Cells Contain a Basic Set of Membrane-enclosed
Organelles
Eukaryotic Cells Contain a Basic Set of Membrane-enclosed
Organelles
Membrane-enclosed Organelles Evolved in Different Ways

• Nuclear membranes and the ER may have evolved through invagination of

the plasma membrane
Membrane-enclosed Organelles Evolved in Different Ways

• Mitochondria are thought to have originated when a prokaryote was engulfed

by a larger eukaryotic cell
Membrane-enclosed organelles import proteins by one of
three mechanisms
Proteins can move between compartments in different ways

“Sorting signals”

“Sorting receptors”
 3 different ways of protein transport between compartments

1. Gated transport
2. Transmembrane transport
3. Vesicular transport
Signal sequences direct proteins to the correct organelle
Gated transport
The nuclear envelope is penetrated by nuclear pores
Proteins Enter the Nucleus Through Nuclear Pores
The nuclear pore complex forms a gate through which molecules
enter or exit from the nucleus

NPC : ~ 125 MDa, composed of 30 different NPC proteins

Electron micrograph of a region of nuclear envelope showing a
side view of two nuclear pore complexes
Electron micrograph showing a face-on view of nuclear pore
complexes
A model for the gated diffusion barrier of the NPC

< 40 kDa
Nuclear Localization Signals (NLSs) direct nuclear proteins to the
nucleus

In general, NLS consists of one or two short sequences are rich in the positively
charged amino acids lysine and arginine.
Proteins bound for the nucleus are actively transported through
nuclear pores
Nuclear Import Receptors bind to both nuclear localization signals and
NPC proteins
Energy supplied by GTP hydrolysis drives nuclear transport
The Ran GTPase imposes directionality on transport through
NPCs
Nuclear export works like nuclear import, but in reverse
Transmembrane transport
The ER is the most extensive membrane network in eukaryotic
cells
 The ER is structurally and functionally diverse

- A central place for both lipid and protein biosynthesis

- A major intracellular store for Ca2+
 The ER is structurally and functionally diverse

- A central place for both lipid and protein biosynthesis

- A major intracellular store for Ca2+
ER membrane contact sites with other organelles
A common pool of ribosomes is used to synthesize all the
proteins encoded by the nuclear genome
An ER signal sequence and an SRP direct a ribosome to the ER
membrane
A SRP directs ER signal sequences to a specific receptor in the
rough ER membrane
A SRP directs ER signal sequences to a specific receptor in the
rough ER membrane
An ER signal sequence is recognized twice: first by an SRP in the
cytosol and then by the pore of the Sec61 complex
The polypeptide chain passes through an aqueous pore in the
translocon Sec61 complex (or translocator)
A single-pass transmembrane protein is integrated into the ER
membrane
A double-pass transmembrane protein uses an internal start-transfer
sequence to integrate into the ER membrane
Proteins are imported into mitochondria in an unfolded form
Translocation into mitochondria depends on signal
sequences and protein translocators
Translocation into mitochondria depends on signal
sequences and protein translocators
Vesicular transport
Vesicles bud from one membrane and fuse with another, carrying
membrane components and soluble proteins between cell
compartments
Vesicular Transport
Vesicle Budding Is Driven by the Assembly of a Protein Coat
Use of Different Coats in Vesicular Traffic
Clathrin molecules form basket-like cages that help shape membrane
into vesicles
Electron micrograph showing numerous clathrin-coated pits and vesicles
budding from the inner surface of the PM
Clathrin-coated vesicles transport selected cargo molecules
Rab proteins and SNAREs help direct transport vesicles to their
target membranes
Rab proteins: monomeric GTPases that control vesicular transport
Following vesicle docking, SNARE proteins can catalyze the fusion
of the vesicle and target membranes
SNAREs Mediate Membrane Fusion
SNAREs Mediate Membrane Fusion
SNARE complexes need to be disassembled before
they can function again
Membrane fusion is more relevant to your life than you might think!

BOTOX

This experiment may not have been done in a scientific way!

How does BOTOX work?
Secretory pathways
 Translocated polypeptide chains fold and assemble in the lumen
of the rough ER

PDI – disulfide bond formation

BiP – reconizes incorrectly folded proteins and keep them from aggregating
Lectins – Calreticulin & calnexin
Many proteins are glycosylated on asparagines in the ER
Oligosaccharides are used as tags to mark the state of protein
folding
Chaperones prevent misfolded or partially assembled proteins
from leaving the ER
Accumulation of misfolded proteins in the ER lumen triggers an
unfolded protein response (UPR)
Improperly folded proteins are exported from the ER and
degraded in the cytosol

ER-associated protein degradation (ERAD)

The golgi apparatus consists of a stack of flattened,
membrane-enclosed sacs
The Golgi apparatus : the major center for protein sorting
The Golgi apparatus : the major center for protein sorting in the
secretory pathway
In secretory cells, the regulated and constitutive pathways of
exocytosis diverge in the trans-Golgi network
Secretory vesicles store insulin in a pancreatic  cells
Endocytosis: Transport into the cell from the PM
Endocytosis: Transport into the cell from the PM
 Endocytosis: Transport into the cell from the plasma membrane

1. Phagocytosis: large particles are ingested via large vesicles

called “phagosomes” (> 250nm in diameter), mainly occurs in
specialized cells – professional phagocytes – macrophages and
neurophils

2. Pinocytosis: fluid and solutes are ingested via relatively small

pinocytic vesicles (~ 100nm in diameter), occurs in most
eukaryotic cells
Specialized phagocytic cells can ingest large particles
Receptor-mediated endocytosis provides a specific route into animal
cells

LDL enters cells via receptor-mediated endocytosis

The fate of receptor proteins following their endocytosis depends on
the type of receptor
A lysosome contains a large variety of hydrolytic enzymes, which
are only active under acidic conditions
Materials destined for degradation in lysosomes follow different
pathways to the lysosome
Several methods can be used to determine whether a labeled
protein bearing a particular signal sequence is transported into a
preparation of isolated organelles
Temperature-sensitive mutants have been used to dissect the protein
secretory pathway in yeasts
Genetics –

How to study genes, whose loss would kill the cell?

Temperature-sensitive mutation :

A conditional mutation that produces the mutant phenotype in one (restrictive

or non-permissive) temperature range and the wild-type phenotype in another
(permissive) temperature range.
Genetics –

How to study genes, whose loss would kill the cell?

Temperature-sensitive mutation :
Genetic screening
How to isolate yeast cells showing phenotype of your interest?

Randy Schekman

Peter Novick
Genetic screening
How to isolate yeast cells showing phenotype of your interest?

A. Wild-type grown at 37℃

B. Mutant grown at 25℃

C. Mutant grown at 37℃ for 1 hr

D. Mutant grown at 37℃ for 3 hr

Genetic screening
How to isolate yeast cells showing phenotype of your interest?

Dense :
Net cell surface
growth stopped
while cell mass
increased

A. Wild-type grown at 37℃

D. Mutant grown at 37℃
Identification of 23 complementation groups required
for yeast secretory pathway

Randy Schekman

Peter Novick
Tagging a protein with GFP allows the resulting fusion protein to
be tracked throughout the cell