10 Protein Sorting and Transport
Chapt. 10: Protein Sorting, Transport:
Endoplasmic Reticulum, Golgi, Lysosomes
Student learning outcomes: organelle functions
• Explain sorting of proteins: free vs. bound ribosomes
• Identify signals on proteins that identify destinations:
Carbohydrates, amino acid sequences, lipids
• Describe complexes that read signals, deliver cargo:
SRP particles, Signal peptidases, chaperones, SNAREs
• State steps taken for proteins destined for secretion,
for a lysosome; state compartments traversed
(compare nucleus); connected by vesicular transport
Fig 10.1 The endoplasmic reticulum (ER)
1. Endoplasmic reticulum (ER); network of
membrane-enclosed tubules and sacs (cisternae);
extends from nuclear membrane through cytoplasm.
•
•
•
•
Membrane is continuous; largest organelle of most cells.
Rough ER is covered by ribosomes.
Transitional ER is where vesicles exit to Golgi apparatus.
Smooth ER no ribosomes, lipid metabolism
Fig. 10.1
Fig 10.2 The secretory pathway
ER and protein processing, secretion:
• Pancreatic acinar cells secrete digestive enzymes
• Newly synthesized proteins briefly labeled with
radioisotopes, chased with nonradioactive
• Proteins located by autoradiography.
Fig. 10.2
Fig 10.3** Overview of protein sorting
**Secretory pathway:
• rough ER → Golgi →
secretory vesicles → cell exterior
Non-secreted proteins:
• free ribosomes →
nucleus, mitochondria,
peroxisome
**Fig. 10.3
The Endoplasmic Reticulum
Ribosomes targeted to ER by signal sequence at
NH2-terminus of polypeptide
• Protein synthesis starts on free ribosomes
Cotranslational translocation:
• Proteins translocated into ER during synthesis on
membrane-bound ribosomes.
Figs. 8.22,27
Posttranslational translocation:
• proteins are translocated into ER after translation completed
on free ribosomes; Hsp70 Chaperone proteins
Fig 10.5 Incorporation of secretory proteins into microsomes
Evidence for signal sequences for ER:
Secretory proteins translated from mRNAs on free
ribosomes are larger than the secreted proteins.
With microsomes, growing polypeptide chains
incorporated into microsomes, signal sequences
removed by proteolytic cleavage.
Fig. 10.5
SDS-PAG analysis
of proteins
synthesized in vitro
vs. secreted (s)
Fig 10.6 The signal sequence of growth hormone
Signal sequences (about 20 amino acids):
• stretch of hydrophobic residues
• usually located at NH2-terminus of polypeptide chain.
Fig. 10.6 Signal sequence of growth hormone
Fig10.8 Cotranslational targeting of secretory proteins to ER
Cotranslational translocation:
• Signal sequences are bound by
signal recognition particle (SRP).
• SRPs have 6 polypeptides,
small cytoplasmic RNA (SRP RNA).
SRP binds ribosome and signal sequence, binds SRP receptor
on ER to make RER
• Ribosome binds translocon;
• Signal sequence inserts
into translocon
• Signal peptidase releases
polypeptide in ER lumen
Fig. 10.7,8 cotranslational
Fig 10.9 Posttranslational translocation of proteins into ER
Posttranslation translocation (more common in yeast):
• Proteins synthesized on free ribosomes
• Signal sequences recognized by receptors on translocon
(not need SRP)
• Hsp70 chaperones keep
polypeptide chains unfolded
so can enter translocon
• Hsp70 chaperone in ER (BiP)
acts as ratchet to pull
polypeptide chain through
Fig. 10.9
Posttranslational
translocation
The Endoplasmic Reticulum
Transmembrane (integral) proteins:
• Proteins destined for incorporation into membranes initially
insert into ER membrane, not release into lumen.
• Transported along secretory pathway as membrane
components rather than soluble proteins
• Membrane-spanning regions of integral membrane proteins
usually α helical regions with ~20-25 hydrophobic amino acids.
• Orientations vary — N or C terminus on cytosolic side
• Some multiple membrane
-spanning regions.
Fig. 10.10
Fig 10.11 Topology of the secretory pathway
** Topology:
Lumens of ER and Golgi
are topologically equivalent
to exterior of cell.
Domains of plasma
membrane proteins
exposed on cell surface
= regions of polypeptide
translocated into ER lumen.
Fig. 10.11
Fig 10.12 Insertion of membrane protein with cleavable signal sequence, single
stop-transfer sequence
Simplest method for Integral protein insertion:
• Signal sequence cleaved by signal peptidase during
translocation, leaves NH2-terminus in ER lumen.
• Translocation halts at stop-transfer sequence;
protein exits laterally and anchors in ER membrane.
Fig. 10.12
Fig 10.13 Insertion of membrane proteins with internal noncleavable signal
sequences
Proteins also anchor in ER membrane by internal
signal sequences not cleaved by signal
peptidase.
• These sequences are
transmembrane α helices
• Exit translocon, anchor
proteins in ER membrane,
in either orientation.
Fig. 10.13
Fig 10.14 Insertion of protein that spans membrane multiple times
Proteins that span the membrane multiple times are
inserted by alternating series of internal signal
sequences, transmembrane stop-transfer sequences.
Fig. 10.14
Fig 10.15 Protein folding in the ER
• Protein folding and processing occur either during
translocation across ER or within ER lumen.
• Lumenal ER proteins assist folding and assembly of
translocated polypeptides
• Hsp70 chaperone BiP binds to unfolded polypeptide chain as
it crosses membrane, helps fold and assemble complexes
• Disulfide (S—S) bond formation facilitated by PDI (protein
disulfide isomerase).
Fig. 10.15
The Endoplasmic Reticulum
Glycosylation:
• (N-linked glycosylation) occurs on specific asparagine (Asn)
residues as protein translocates into ER.
• Oligosaccharide is synthesized on lipid (dolichol) carrier.
• Glycosylation prevents
protein aggregation
in ER, provides
signals for sorting.
Fig. 10.16
Fig 10.17 Addition of GPI anchors
Glycosylphosphatidylinositol (GPI) anchors
• Glycolipids attach some proteins to plasma membrane
GPI anchors assemble in ER membrane, add to C-terminal Asn
• GPI-anchored proteins are
transported as membrane
components via secretory path.
• Topology within ER dictates
they are exposed outside of cell.
Fig. 8.36
GPI anchor Thy-1
Fig. 10.17
Fig 10.18 Glycoprotein folding by calreticulin
Chaperones and sensors in ER identify misfolded
proteins, divert them to degradation pathway.
• Ex. chaperone calreticulin assists glycoproteins folding
• Protein folding sensor
passes correctly folded
glycoproteins on to
transitional ER.
Fig. 10.18
The Endoplasmic Reticulum
Unfolded protein response (UPR)
BiP plays role as sensor of general state of protein folding.
• If excess of unfolded proteins accumulates, BiP initiates UPR:
• Includes general inhibition of protein synthesis, increased
expression of chaperones, increase in degradation of mRNAs.
Fig. 10.19
2. Smooth Endoplasmic Reticulum
2. Lipid synthesis occurs on smooth ER
• Eukaryotic membranes 3 types of lipids:
phospholipids, glycolipids, and cholesterol.
• Phospholipids are synthesized on cytosolic side of ER from
water-soluble precursors (glycerol, fatty acids) (Fig. 10.20).
Fig. 10.20*
The Endoplasmic Reticulum
*Flippase moves phospholipids across membrane:
• Synthesis of new phospholipids on cytosolic side keeps
hydrophobic fatty acid chains buried in membrane.
• Transfer to other half requires
passage of polar head groups
through membrane,
facilitated by membrane flippases
• Ensures even growth of both sides
of phospholipid bilayer.
Fig. 10.21
The Endoplasmic Reticulum
Smooth ER: synthesis of cholesterol, ceramide.
• Steroid hormones are synthesized from cholesterol in the ER
• Ceramide is converted to glycolipids or sphingomyelin in the
Golgi apparatus.
• Smooth ER is abundant in
cells with active
lipid metabolism.
• Smooth ER in liver has
enzymes to detoxify
lipid-soluble compounds
Fig. 10.22
The Endoplasmic Reticulum – Vesicles **
Vesicles export proteins and phospholipid molecules
from ER, bud from transitional ER, move through ERGolgi intermediate compartment, and then to Golgi.
• Proteins in lumen of one
organelle bud into transport
vesicles, release to lumen of
recipient organelle by vesicle
fusion.
• Membrane proteins and lipids
are transported in similar way;
topological orientation is
maintained.
Fig. 10.23*
The Endoplasmic Reticulum
ER Export: most proteins entering transitional ER are
marked by sequences that signal export from ER.
• Transmembrane proteins often di-acidic or di-hydrophobic
amino acid signals in cytosolic domains.
• GPI-anchored proteins are marked for export by GPI anchors.
Fig. 10.24
The Endoplasmic Reticulum
Return to the ER:
• Some proteins must stay in ER (BiP, signal peptidase, etc).
• Target sequence
(KDEL or KKXX)
at carboxy terminus
directs retrieval from
ERGIC or Golgi complex
via recycling path.
Fig. 10.25
The Golgi Apparatus
3. the Golgi apparatus, or Golgi complex:
• Proteins from ER are processed and sorted for transport: to
endosomes, lysosomes, plasma membrane, or secretion.
• Most glycolipids and sphingomyelin are made in Golgi,
• The Golgi is composed of flattened membrane-enclosed sacs
(cisternae) and associated vesicles:
• cis Golgi network
receives molecules from ERGIC
• medial and trans Golgi stacks
most modifications here
• trans Golgi network
sorting and distribution
Fig. 10.26
The Golgi Apparatus
Mechanism of protein
movement is not certain;
they may be carried in
cisternae, which gradually
mature, move through Golgi
in cis to trans direction.
Transport vesicles return Golgi
resident proteins for reuse.
Fig. 10.27
The Golgi Apparatus
Glycoproteins are processed in the Golgi:
• Glycosyltransferases add different sugar residues;
glycosidases remove sugar residues
• modification of N-linked oligosaccharides added in ER.
• Mannose residues are removed; N-acetylglucosamine,
galactose, and sialic acid residues are added.
• Proteins emerge with different N-linked oligosaccharides
Fig. 10.28 Golgi
modifications
The Golgi Apparatus
*Lysosomal proteins: N-linked oligosaccharides
modified by mannose phosphorylation.
• Enzyme recognizes structural signal (shape) on folded
lysosomal proteins: signal patch, not a linear aa sequence.
• Lysosomal protein binds mannose-6-phosphate receptor in
trans Golgi, which transports it to endosomes and lysosomes.
Fig. 10.29
The Golgi Apparatus
Glycolipids
• synthesized from ceramide by addition of carbohydrates.
Sphingomyelin (nonglycerol
phospholipid in membranes)
• made by transfer of
phosphorylcholine group
from phosphatidylcholine
to ceramide.
Fig. 10.30
Made in smooth ER
Fig 10.31 Transport from the Golgi apparatus
trans Golgi sorts, packages molecules in transport vesicles
Proteins staying in the Golgi bind the membrane; signals
preventing packaging and transport.
Intracellular destinations:
late endosome, lysosome,
vacuole (yeast, plants lack lysosome)
Transport to cell surface :
– Direct transport
– Recycling endosomes
– Regulated secretory paths
(vesicles)
Fig. 10.31
The Golgi Apparatus
Regulated secretion pathways:
• release of hormones from endocrine cells
• neurotransmitters from nerve cells.
• digestive enzymes from pancreatic cells
• These proteins have patch signals
recognized by cargo receptors.
• released in secretory vesicles:
contents stored until signals direct fusion
with plasma membrane.
Fig. 10.31
Fig 10.32 Transport to the plasma membrane of polarized cells
Polarized epithelial cells have plasma membrane divided into
apical domain, and basolateral domain:
each has specific proteins.
Proteins leaving trans Golgi
selectively packaged,
transported to correct domain.
Fig. 10.32
The Mechanism of Vesicular Transport
Selectivity of vesicular transport
is key to functional organization of a cell.
• Vesicles must recognize and fuse only with appropriate target
membrane.
• Understanding mechanisms that control vesicular transport is
major area of research in cell biology.
• Includes secretion and uptake of molecules (Ch. 13)
Experimental approaches to understanding transport include:
Isolation of yeast mutants defective in transport and sorting (sec mutants)
Reconstitution of vesicular transport in cell-free systems
Biochemical analysis of synaptic vesicles (neurons)
Tracing path of specific GFP fusion proteins through secretory network
Proteomic analysis of secretory compartments
The Mechanism of Vesicular Transport
Cargo selection, coat proteins, vesicle budding
Transport vesicles with
secretory proteins have
cytosolic coat proteins.
Coats assemble as vesicle buds;
are removed in cytosol before
vesicle reaches its target.
Vesicles fuse with target
membrane, empty cargo, and
insert their membrane proteins
into target membrane.
Fig. 10.34
The Mechanism of Vesicular Transport
Three families of vesicle coat proteins



COPII-coated vesicles carry
secretory proteins from ER to
ERGIC to Golgi apparatus
COPI-coated vesicles bud from
ERGIC or Golgi, carry cargo
backwards, return proteins to
earlier compartments.
*Clathrin-coated vesicles
transport in both directions
between trans Golgi network,
endosomes, lysosomes, and
plasma membrane.
Fig. 10.35
Fig 10.37 Incorporation of lysosomal proteins into clathrin-coated vesicles
Clathrin-coated vesicles require
•
•
•
•
clathrin, GTP-binding protein ARF1,
and adaptor proteins
Clathrin assembles into basketlike lattice
that distorts membrane, starts bud
Proteins targeted for lysosomes have
Mannose-6-PO4, binds Mannose-6-PO4
receptors in trans Golgi membrane
Mannose-6-PO4 receptors span the Golgi
membrane, binding sites for cytosolic
adaptor proteins, which bind clathrin
Clathrin-coated vesicles have many
destinations; different adaptors
Fig. 10.37
The Mechanism of Vesicular Transport
SNARE hypothesis:
Vesicle fusion occurs by interactions between pairs of
transmembrane proteins, called SNAREs, on
vesicle and target membranes (v-SNAREs, t-SNAREs).
SNARE-SNARE pairing provides energy to bring two
bilayers close enough to destabilize, permit fusion
Rab family of small GTP-binding proteins plays key
role in vesicle budding and fusion:
• Mark different organelles and transport vesicles, so need to
be localized on correct membrane for specificity
Rab proteins have hydrocarbon prenyl tail to insert in membrane
Fig 10.39 Vesicle fusion SNARE hypothesis
Rab/GTP on transport vesicle interacts with effector
proteins and v-SNAREs to assemble pre-fusion complex
• Different Rab protein on
target membrane
organizes other effector
proteins and t-SNAREs.
• Effector proteins link
membranes by proteinprotein interactions
(tethering).
• After fusion, protein
complex (NSF/SNAP
complex) disassembles
SNAREs for reuse
Fig. 10.39 SNAREs
Fig 10.41 Electron micrograph of lysosomes, mitochondria in mammalian cell
4. Lysosomes
• Membrane-enclosed organelles with ~ 50 acid hydrolases
•
•
Enzymes break down diverse macromolecules
Digestive system of the cell, recycles organelles also.
Fig. 10.41 lysosomes (→)
and mitochondria
Lysosomes and disease
Lysosomal storage diseases:
• Mutations in genes encoding lysosomal enzymes:
• Undegraded material accumulates,
• Impairs macrophages, neural cells.
• Ex. Gaucher disease is glucocerebrosidase defect
• Treat with enzyme replacement therapy
Lysosomes
• Most lysosomal enzymes are acid
hydrolases: active at pH 5 in lysosomes,
not cytoplasm (pH 7.2).
• Acid activity protects against
uncontrolled digestion of cell contents;
• If lysosome membrane breaks, acid
hydrolases are inactive in cytosol.
• To maintain acidic pH, a proton pump
in lysosomal membrane actively
transports protons
Fig. 10.42
Fig 10.43 Endocytosis and lysosome formation
Lysosomes digest material taken up from
outside by endocytosis. (Chapt. 13)
Endosomes are intersection between
secretory and endocytic pathway
Endocytosis and lysosome formation:
Early endosomes
Separate molecules for recycling (e.g. receptors)
from those for degradation
Molecules to be recycled go to recycling endosomes
and back to plasma membrane.
Late endosomes receive lysosomal
enzymes from trans Golgi network,
can fuse or mature into lysosomes
Fig. 10.43
Lysosomes
• Phagocytosis:
• Specialized cells such as macrophages take up, degrade
large particles (bacteria, cell debris, aged cells.
• Particles in phagocytic vacuoles (phagosomes), fuse with
lysosomes to become phagolysosomes.
• Autophagy:
• turnover of
cell contents
old organelles
Fig. 10.44
Review
Review questions:
1. Describe experimental evidence for secretory path:
ER -> Golgi -> secretory vesicle -> secreted
protein
3. Compare/ contrast co-translational with posttranslational translocation of polypeptides into ER
5. Why are carbohydrate groups of glycoproteins
always exposed on the surface of the cell?
Review
6. What would be effect of mutating KDEL sequence
of resident ER protein like BiP? How would this
differ from mutating the KDEL receptor protein?
7. How is a lysosomal protein targeted to lysosome?
What about adding a lysosome-targeting signal
patch on protein that is normally cytosolic?
9. What processes result in glycolipids and
sphingomyelin being found in outer – but not innerhalf of plasma membrane bilayer?