10

Protein Sorting and Transport:

The Endoplasmic Reticulum, Golgi Apparatus,
and Lysosomes
10 Introduction

• In addition to the presence of a nucleus,

eukaryotic cells are distinguished from prokaryotic
cells by the presence of membrane-enclosed
organelles within their cytoplasm.
• Many proteins destined for the endoplasmic
reticulum, the Golgi apparatus, lysosomes, the
plasma membrane, and secretion from the cell
are synthesized on ribosomes that are bound to
the membrane of the endoplasmic reticulum.
10 The Endoplasmic Reticulum

• The endoplasmic reticulum, or ER, is a network of

membrane-enclosed tubules and sacs that
extends from the nuclear membrane throughout
the cytoplasm.
• The rough ER is covered by ribosomes on its
outer surface and is involved in protein
metabolism.
10.1 The endoplasmic reticulum (ER)
10 The Endoplasmic Reticulum

• The transitional ER is involved in protein

processing and is where vesicles exit to the Golgi
apparatus.
• The smooth ER is involved in lipid, rather than
protein, metabolism and is not associated with
ribosomes.
10 The Endoplasmic Reticulum and Protein Secretion

• The endoplasmic reticulum plays a key role in

protein processing and sorting.
• Secretory vesicles carry radiolabeled proteins
from the Golgi apparatus to the cell surface, then
fuse with the plasma membrane to release their
contents outside of the cell.
10.2 The secretory pathway
10 The Endoplasmic Reticulum and Protein Secretion

• The secretory pathway consists of the rough ER

→ Golgi → secretory vesicles → cell exterior.
• The entrance of proteins into the ER represents a
major branch point for the traffic of proteins within
eukaryotic cells.
10.3 Overview of protein sorting
10 Targeting Proteins to the Endoplasmic Reticulum

• Proteins can be translocated into the ER either

during their synthesis on membrane-bound
ribosomes or after their translation has been
completed on free ribosomes in the cytosol.
• A signal sequence targets ribosomes, engaged in
the synthesis of proteins that are destined for
secretion, to the endoplasmic reticulum.
10.4 Isolation of rough ER
10 Targeting Proteins to the Endoplasmic Reticulum

• Microsomes are small vesicles formed from the

endoplasmic reticulum when cells are disrupted.
• The signal for ribosome attachment to the ER
might be an amino acid sequence near the amino
terminus of the growing polypeptide chain.
10.5 Incorporation of secretory proteins into microsomes
10 Targeting Proteins to the Endoplasmic Reticulum

• It has been hypothesized and substantiated that

an amino terminal leader sequence targets the
polypeptide chain to the microsomes and is then
cleaved by a microsomal protease.
• The mechanism by which secretory proteins are
targeted to the ER during their translation consists
of signal sequences that span about 20 amino
acids.
10.6 The signal sequence of growth hormone
10 Targeting Proteins to the Endoplasmic Reticulum

• Signal recognition particles, or SRPs, recognize

and bind to signal sequences as the latter emerge
from the ribosome.
• Small cytoplasmic RNAs, or srpRNAs, are
components of SRP.
10.7 Structure of the SRP
10 Targeting Proteins to the Endoplasmic Reticulum

• SRP receptors are proteins on the membrane of

the endoplasmic reticulum that bind the signal
recognition particle, SRP.
• A translocon is a membrane channel through
which polypeptide chains are transported into the
endoplasmic reticulum.
10.8 Cotranslational targeting of secretory proteins to the ER
10 Targeting Proteins to the Endoplasmic Reticulum

• Yeast and mammalian translocon proteins are

closely related to the plasma membrane proteins
that translocate secreted polypeptides in bacteria.
• Signal peptidase cleaves the signal sequence and
releases it into the lumen of the ER.
10 Targeting Proteins to the Endoplasmic Reticulum

• Many proteins in yeast are targeted to the ER

after their translation is complete rather than
being transferred into the ER during synthesis on
membrane-bound ribosomes.
10.9 Posttranslational translocation of proteins into the ER
10 Insertion of Proteins into the ER Membrane

• Proteins destined for secretion from the cell or

residence within the lumen of the ER, Golgi
apparatus, endosomes, or lysosomes are
translocated across the ER membrane and
released into the lumen of the ER.
• Integral membrane proteins are embedded in the
membrane by hydrophobic sequences that span
the phospholipid bilayer.
10.10 Orientations of membrane proteins
10 Insertion of Proteins into the ER Membrane

• The lumen of the ER is topologically equivalent to

the exterior of the cell, so the domains of plasma
membrane proteins that are exposed on the cell
surface correspond to the regions of polypeptide
chains that are translocated into the ER lumen.
• The most straightforward mode of insertion into
the ER membrane results in the synthesis of
transmembrane proteins oriented with their
carboxy termini exposed to the cytosol.
10.11 Topology of the secretory pathway
10.12 Membrane protein with a cleavable signal sequence and a single stop-transfer sequence
10 Insertion of Proteins into the ER Membrane

• Proteins can also be anchored in the ER

membrane by internal signal sequences that are
not cleaved by signal peptidase.
• Proteins that span the membrane multiple times
are thought to be inserted as a result of an
alternating series of internal signal sequences and
transmembrane stop-transfer sequences.
10.13 Insertion of membrane proteins with internal noncleavable signal sequences (Part 1)
10.13 Insertion of membrane proteins with internal noncleavable signal sequences (Part 2)
10.14 Insertion of a protein that spans the membrane multiple times
10 Insertion of Proteins into the ER Membrane

• Most transmembrane proteins destined for other

compartments in the secretory pathway are
delivered to them in transport vesicles.
10 Protein Folding and Processing in the ER

• For proteins that enter the secretory pathway,

many of these events occur either during
translocation across the ER membrane or within
the ER lumen.
• After proteins are translocated across the ER
membrane as unfolded polypeptide chains, they
fold into their three-dimensional conformations,
assisted by molecular chaperones.
10.15 Protein folding in the ER
10 Protein Folding and Processing in the ER

• Protein disulfide isomerase is located in the ER

lumen and is an enzyme that facilitates disfulfide
bond formation.
• Glycosylphosphatidylinositol, or GPI anchors, are
membrane-anchoring glycolipids that contain
phosphatidylinositol.
10.16 Protein glycosylation in the ER
10.17 Addition of GPI anchors
10 Quality Control in the ER

• Many proteins synthesized in the ER are rapidly

degraded, primarily because they fail to fold
correctly; others reside in the ER for several hours
while they are properly folded.
• The unfolded protein response is a cellular stress
reponse in which an excess of unfolded proteins
in the endoplasmic reticulum leads to general
inhibition of protein synthesis, increased
expression of chaperones, and increased
proteasome activity.
10.18 Glycoprotein folding by calnexin
10.19 Unfolded protein response (Part 1)
10.19 Unfolded protein response (Part 2)
10 The Smooth ER and Lipid Synthesis

• Because they are extremely hydrophobic,

membrane lipids are synthesized in association
with already existing cellular membranes rather
than in the aqueous environment of the cytosol.
• The membranes of eukaryotic cells are composed
of three main types of lipids: phospholipids,
glycolipids, and cholesterol.
10.20 Synthesis of phospholipids
10.20 Synthesis of phospholipids (Part 1)
10.20 Synthesis of phospholipids (Part 2)
10 The Smooth ER and Lipid Synthesis

• The synthesis of phospholipids on the cytosolic

side of the ER membrane allows the hydrophobic
fatty acid chains to remain buried in the
membrane while membrane-bound enzymes
catalyze their reactions with water-soluble
precursors in the cytosol.
• Flippases are membrane proteins that catalyze
the rapid translocation of phospholipids across the
ER membrane resulting in even growth of both
halves of the bilayer.
10.21 Translocation of phospholipids across the ER membrane
10 The Smooth ER and Lipid Synthesis

• In addition to the role in synthesis of the glycerol

phospholipids, the ER also serves as the major
site of synthesis of two other membrane lipids:
cholesterol and ceramide.
• Smooth ER is abundant in cell types that are
particularly active in lipid metabolism.
10.22 Structure of cholesterol and ceramide
10 Export of Proteins and Lipids from the ER

• Both proteins and phospholipids travel along the

secretory pathway in transport vesicles, which
bud from the membrane of one organelle and
then fuse with the membrane of another.
• Most proteins that enter the transitional ER move
through the ER-Golgi intermediate compartment
and on to the Golgi.
10.23 Vesicular transport from the ER to the Golgi
10.24 ER export signals
10 Export of Proteins and Lipids from the ER

• If proteins that function within the ER are allowed

to proceed along the secretory pathway, they will
be lost to the cell.
• KDEL and KKXX signals do not prevent ER
proteins from being packaged into vesicles and
carried to the Golgi; instead they cause these ER
resident proteins to be selectively retrieved from
the ER-Golgi intermediate compartment or the
Golgi complex and returned to the ER via a
recycling pathway.
10.25 Retrieval of resident ER proteins
10 The Golgi Apparatus

• The Golgi apparatus, or Golgi complex, functions

as a factory in which proteins received from the
ER are further processed and sorted for transport
to their eventual destinations—endosomes,
lysosomes, the plasma membrane, or secretion
from the cell.
• In plant cells, the Golgi apparatus further serves
as the site at which the complex polysaccharides
of the cell wall are synthesized.
10 Organization of the Golgi

• In most cells, the Golgi is composed of flattened

membrane-enclosed sacs and associated
vesicles.
• The cis Golgi network is the region of the Golgi
apparatus at which proteins enter from the
endoplasmic reticulum.
10.26 Electron micrograph of a Golgi apparatus
10 Organization of the Golgi

• The Golgi stack consists of the compartments of

the Golgi apparatus within which most metabolic
activities take place.
• The trans Golgi network is the Golgi compartment
within which proteins are sorted and packaged to
exit the Golgi apparatus.
• The mechanism by which proteins move through
the Golgi apparatus has still not been established
and is an area of controversy among cell
biologists.
10.27 Regions of the Golgi apparatus
10 Protein Glycosylation within the Golgi

• Protein processing within the Golgi involves the

modification and synthesis of the carbohydrate
portions of glycoproteins.
• N-linked oligosaccharides are processed within
the Golgi apparatus in an ordered sequence of
reactions.
10.28 Processing of N-linked oligosaccharides in the Golgi
10 Protein Glycosylation within the Golgi

• A glycosyltransferase is an enzyme that adds

sugar residues to its substrate.
• A glycosidase is an enzyme that removes sugar
residues from its substrate.
• The processing of the N-linked oligosaccharide of
lysosomal proteins differs from that of secreted
and plasma membrane proteins.
10.29 Targeting of lysosomal proteins by phosphorylation of mannose residues
10 Protein Glycosylation within the Golgi

• Mannose-6-phosphate residues are what is left on

the N-linked oligosaccharide after N-
acetylglucosamine is removed.
• Signal patches are recognition determinants
formed by the three-dimensional folding of a
polypeptide chain.
10 Lipid and Polysaccharide Metabolism in the Golgi

• In addition to its activities in processing and

sorting glycoproteins, the Golgi apparatus
functions in lipid metabolism—in particular, in the
synthesis of glycolipids and sphingomyelin.
• Sphingomyelin is synthesized on the lumenal
surface of the Golgi, but glucose is added to
ceramide on the cytosolic side.
• In plant cells, the Golgi apparatus has the
additional task of serving as the site where
complex polysaccharides of the cell wall are
synthesized.
10.30 Synthesis of sphingomyelin and glycolipids
10 Protein Sorting and Export from the Golgi
Apparatus

• Proteins as well as lipids and polysaccharides are

transported from the Golgi apparatus to their final
destinations through the secretory pathway.
• The constitutive secretory pathway, which
operates in all cells, leads to continual
unregulated protein secretion.
10.31 Transport from the Golgi apparatus
10 Protein Sorting and Export from the Golgi
Apparatus

• The apical domain is the exposed free surface of

a polarized epithelial cell.
• The basolateral domain is the surface region of a
polarized epithelial cell that is in contact with
adjacent cells or the extracellular matrix.
• The apical membrane of intestinal epithelial cells
faces the lumen of the intestine and is specialized
for the efficient absorption of nutrients; the
remainder of the cell is covered by the basolateral
membrane.
10.32 Transport to the plasma membrane of polarized cells
10 Protein Sorting and Export from the Golgi
Apparatus

• A vacuole is a large membrane-enclosed sac in

the cytoplasm of eukaryotic cells.
 In
plant cells, vacuoles function to store
nutrients and waste products, to degrade
macromolecules, and to maintain turgor
pressure.
10.33 A plant cell vacuole
10 The Mechanism of Vesicular Transport

• Vesicular transport is a major cellular activity,

responsible for molecular traffic between a variety
of specific membrane-enclosed compartments.
• The selectivity of such transport is key to
maintaining the functional organization of the cell.
10 Experimental Approaches to Understanding
Vesicular Transport

• Progress toward elucidating the molecular

mechanisms of vesicular transport has been
made by several distinct experimental
approaches.
• Biochemical studies of vesicular transport using
reconstituted systems have complemented
genetic studies and have enabled the direct
isolation of transport proteins from mammalian
cells.
• Synaptic vesicles are secretory vesicles that
release neurotransmitters at a synapse.
10 Cargo Selection, Coat Proteins, and Vesicle
Budding

• Most transport vesicles that carry secretory

proteins from the ER to the Golgi and from the
Golgi to other targets are coated with cytosolic
coat proteins and thus are called coated vesicles.
• The formation of coated vesicles is regulated by
small GTP-binding proteins related to Ras and
Ran.
10.34 Formation and fusion of a transport vesicle
10 Cargo Selection, Coat Proteins, and Vesicle
Budding

• COP-coated vesicles are transport vesicles

coated with COP I or COP II.
• COP I and COP II are two proteins other than
clathrin that coat transport vesicles.
• Clathrin is a protein that coats the cytoplasmic
surface of cell membranes and assembles into
basketlike lattices that drive vesicle budding.
10.35 Initiation of a clathrin-coated vesicle by ARF1
10.36 Incorporation of lysosomal proteins into clathrin-coated vesicles
10.36 Incorporation of lysosomal proteins into clathrin-coated vesicles (Part 1)
10.36 Incorporation of lysosomal proteins into clathrin-coated vesicles (Part 2)
10 Vesicle Fusion

• The fusion of a transport vesicle with its target

involves two types of events.
• The SNARE hypothesis states that vesicle fusion
is mediated by pairs of transmembrane proteins,
or SNAREs, on the vesicle and target
membranes.
• The Rab family of small GTP-binding proteins
plays a key role in vesicular transport.
10 Vesicle Fusion

• Individual Rab proteins or combinations of Rab

proteins mark different organelles and transport
vesicles, so their localization on the correct
membrane is key to establishing the specificity of
vesicular transport.
• To initiate transport vesicle fusion, Rab/GTP on
the transport vesicle interacts with effector
proteins and v-SNAREs to assemble a pre-fusion
complex.
10.37 Delivery of Rab to a membrane
10.38 Vesicle fusion
10 Vesicle Fusion

• Membrane fusion is a general process that occurs

whenever a transport vesicle fuses with a target
membrane.
10.39 Exocyst assembly and vesicle targeting
10 Lysosomes

• Lysosomes are membrane-enclosed organelles

that contain an array of enzymes capable of
breaking down all types of biological polymers.
10.40 Electron micrograph of lysosomes and mitochondria in a mammalian cell
10 Lysosomal Acid Hydrolases

• Lysosomal storage diseases are a family of

diseases characterized by the accumulation of
undegraded material in the lysosomes of affected
individuals.
• Most lysosomal enzymes are acid hydrolases,
which are active at the acidic pH that is
maintained within lysosomes but not at the neutral
pH characteristic of the rest of the cytoplasm.
10.41 Organization of the lysosome
10 Endocytosis and Lysosome Formation

• Endocytosis is the uptake of extracellular material

in vesicles formed from the plasma membrane.
• Endosome formation represents an intersection
between the secretory pathway through which
lysosomal proteins are processed, and the
endocytic pathway through which extracellular
molecules are taken up at the cell surface.
• Late endosomes mature into lysosomes as they
acquire a full complement of acid hydrolases,
which digest the molecules originally taken up by
endocytosis.
10.42 Endocytosis and lysosome formation
10.42 Endocytosis and lysosome formation (Part 1)
10.42 Endocytosis and lysosome formation (Part 2)
10 Phagocytosis and Autophagy

• In addition to degrading molecules taken up by

endocytosis, lysosomes digest material derived
from two other routes—phagocytosis and
autophagy.
• In phagocytosis, specialized cells, such as
macrophages, take up and degrade large
particles, including bacteria, cell debris, and aged
cells that need to be eliminated from the body.
• Phagosomes are phagocytic vacuoles that take in
these large particles.
10.43 Lysosomes in phagocytosis and autophagy
10 Phagocytosis and Autophagy

• Phagosomes fuse with lysosomes and become

phagolysosomes.
• The process of autophagy takes place in
lysosomes and is the gradual turnover of the cell’s
own components.
• An autophagosome is a vesicle containing internal
organelles enclosed by fragments of the
endoplasmic reticulum membrane. The
autophagosome fuses with a lysosome and its
contents are digested.