Transcript Lecture 9-10 Slides

BIOL 200 (Section 921)
Lecture # 9, 10
June 29/30, 2006
Readings
• ECB (2nd ed.) Chapter 15 (Whole chapter):
Introduction, pp. 496-501; Protein Sorting pp.
502-504; Protein targeting to the Endoplasmic
Reticulum, pp. 505-512; Vesicular Transport, pp.
512 – 516; Secretory Pathway, pp. 516 – 523;
Endocytotic Pathway, pp. 523 -529. Questions:
15-3, 15-4, 15-6 to 15-9, 15-12, 15-13, 15-15, 1516, 15-18, 15-19, 15-21).
Learning Objectives
I. Protein Targeting - Nucleus, Mitochondria,
Chloroplasts and ER.
• To explain the nature of signals and sorting
• To explain the function of coat proteins and the signals carrried by
vesicular conponents.
• To understand the role and importance of the signal recognition
particle and the SRP Receptor in the targeting of proteins to the
endoplasmic reticulum.
• To explain how single and multiple pass membrane proteins are
inserted into the membrane through use of signal sequences, start
transfer sequences and stop transfer sequences.
II. Golgi, Vesicle Transport, Endocytosis, Exocytosis.
• Become familiar with the structure and overall function of the Golgi
apparatus
• Understand how vesicles are formed and targeted.
• To be able to trace a molecule through each of these pathways
describing all of the structures and process through which they pass.
Eukaryotes have many membrane
bound compartments
pancreas cell
15_02_cell_intestine.jpg
How are proteins transported intra- and inter-cellularly?
01_24_Organelles.jpg
15_05_import_proteins.jpg
Signal sequences direct proteins to specific organelles
15_06_Signal_sequence.jpg
Transport of proteins through nuclear
pores
• Active transport –
requires GTP
hydrolysis
• Prospective nuclear
proteins have a
nuclear localization
signal (NLS): -ProLys-Lys-Lys-ArgLys-Val-
Protein transport in mitochondria
15_10_unfolded_imprt.jpg
Chaperone proteins pull the proteins
across the membranes and refold them
Inside the organelles
Structure of ER: (A) Green fluorescent protein fused to
ER resident protein; (B) TEM of a thin section
15_11_ER.jpg
15_12_pool_ribosomes.jpg
Synthesis of cytosolic and
ER-bound proteins
An ER signal sequence, a signal recognition particle
(SRP) and an SRP receptor are required for transport
15_13_ER_signal_SRP.jpg
of a protein
into the ER
A soluble protein enters the ER lumen via the protein
translocation channel [Fig. 15-14]
15_14_enters_lumen.jpg
A single-pass transmembrane protein uses two hydrophobic signal sequences:
a N-terminal start transfer sequence and a stop transfer sequence
15_15_into_ER_membr.jpg
A double-pass ER transmembrane protein uses an internal start-transfer
sequence to integrate into the ER membrane [Fig. 15-16]
15_16_double_pass.jpg
A multipass ER transmembrane protein uses many pairs of start and stop sequences
01_25_endocytosis exoc.jpg
Endocytosis
Vesicular transport
- Endocytosis
- Exocytosis (secretion)
Exocytosis
(secretion)
QUIZ: Name the compartments of the secretory
and endocytic pathways [Vesicular transport]
6
4
3
5
2
Fig. 15-17
1
Secretory and Endocytic pathways [Fig. 15.24]
lysosome
endosome
15_17_Vesicles_bud.jpg
Different vesicle coats drive budding on different
compartments [Table 15-4]
Table 15-4: Different vesicle coats drive
budding on different compartments
Type of coat Coat
proteins
Clathrin
Clathrin +
adaptin 1
Donor
Target
Trans-Golgi
Clathrin
PM
Lysosome
(via
endosome)
endosome
ER
Cis-Gol gi
Golgi
Golgi, ER
COP-II
COP-I
Clathrin +
adaptin 2
COP
proteins
COP
proteins
15_18_Clathrin_EM.jpg
Clathrin protein coat molecules
form basketlike cages that help
shape membranes into vesicles
[Fig. 15.24]
Clathrin protein Lattices
[Becker]
Clathrin triskelions
[Becker]
Transport of specific proteins by clathrin-coated vesicles
15_19_Clathrin_vesicle.jpg
GTP-binding
The specificity of transport vesicles for their target membranes depends on
specific marker proteins, called SNARES
15_20_SNAREs.jpg
SNARE proteins play a central role in membrane
fusion [Fig. 15-21]
15_21_membr_fusion.jpg
1.The vesicle is recognized
by a coiled-coil tethering
protein and a multisubunit
tethering complex
4. Binding of the NSF and
SNAPs proteins promotes
dissociation of SNARE
complexes
2. A RabGTPase bound to
the vesicle stimulates
formation of a stable
complex of one v-SNARE
and 3 t-SNARE helices
3. The v-SNARE/t-SNARE
interaction promotes
Fusion of membranes
The SNARE hypothesis for transport
vesicle targeting and fusion [Becker]
What kind of chemical
modifications does a protein
undergo during vesicular
transport?
Early glycosylation (N-linked) of proteins in the ER [Fig. 15-22]
15_22_glycosylated_ER.jpg
Chaperones prevent misfolded or partially
assembled Proteins from leaving the ER
15_23_Chaperones.jpg
[Fig. 15-23]
Cystic fibrosis: a genetic disease in which a plasma membrane transport protein
is slightly misfolded. It would still function if it reached the plasma membrane. But
it is retained and degraded in the RER.
Functions of Endoplasmic reticulum
1. controls calcium levels in cytoplasm by acting as a
calcium store (cell signaling, muscle contraction)
2. site of membrane lipid biosynthesis (sterols and
phospholipids).
3. entry point for proteins into the secretory pathway
4. site of post-translational modifications of proteins, eg
protein disulfide isomerase forms disulfide bonds here,
glycosylation starts here.
5. site of protein folding by chaperone proteins such as BIP
(binding protein) which prevent hydrophobic domains of
proteins from aggregating and promotes proper folding.
6. quality control checks for proteins (proteins are not
exported from ER if they are properly assembled)
one Golgi stack [Fig. 15-24]
Golgi: stacks of cisternae
Fig. 1-23
Each stack=a dictyosome,
consists of cisternae
(singular, cisterna)
CIS
TRANS
TGN
cis/trans polarity
Models of Golgi Function
There are two competing theories:
Cisternal progression model:
New cisternae form continuously
from ER vesicles.
Cisternae move through the
stack from cis to trans and finally
break up
into transport vesicles at the
trans face.
Vesicle transport model:
Cisternae remain fixed. Both
membrane and content move
from the cis to the trans
cisternae in transport vesicles.
Glu
Man
NGln
In ER,
oligosaccharide
added, modified.
In Golgi, new sugars
added to oligosaccharide
Protein modifications
in Golgi [Fig. 12-6
from Becker]
Predict the location of enzymes,
galactosyl transferase and
sialic acid transferase
The regulated and constitutive pathways of exocytosis [Fig. 15-28]
15_28_trans_Golgi_net.jpg
Targeting of soluble
lysosomal enzymes
to endosomes and
lysosomes by a
Mannose 6-phosphate
tag [Becker Fig. 12-9]
Protein targeting to lysosomes
1. Proteins destined for lysosomes have a special
targeting signal.
2. They are all glycosylated and some of the
mannose residues in the attached
oligosaccharides are phosphorylated to form
mannose-6-phosphate.
3. Mannose-6-phosphate is the targeting signal
for lysosomal proteins.
4. The trans Golgi network contains a special
mannose-6-phosphate receptor that binds to and
results in the concentration of proteins carrying
oligosaccharides bearing mannose-6-phosphate
into vesicles that are destined for lysosomes.
Proteolytic cleavage of proteins as part
of processing in secretory granules
1. The later stages in processing of many secreted
proteins (e.g. digestive enzymes) involves
proteolytic cleavage of a large proprotein to
produce a smaller active protein.
2. This typically occurs in secretory granules as
they move away from the trans Golgi network.
3. These cleavages are carried out by specific
endoproteases (enzymes that cleave polypeptides
at sites within the chain).
Increased blood glucose level regulates exocytosis
of insulin from pancreatic β cells [Fig. 15-29]
Endocytic pathways
1.
2.
3.
4.
Phagocytosis
Pinocytosis
Receptor-mediated endocytosis
Intracellular digestion in lysosomes
Phagocytosis: A white blood cell ingests a bacterium [Fig. 15-30]
15_30_white_bloodcell.jpg
Phagocytosis: A macrophage engulfs two red blood cells [Fig. 15-31]
15_31_macrophage.jpg
Autophagic digestion of mitochondria [Becker]
LDL enters cells via receptor-mediated endocytosis [Fig. 15-32]
15_32_LDL_enters.jpg
Receptor mediated endocytosis [Becker Fig. 12-15; ECB Fig. 15-33]
A lysosome contains hydrolytic enzymes and a H+ pump [Fig. 15-34]
15_34_lysosome.jpg
Different pathways leading to intracellular digestion in lysosomes [15-35]
15_35_path_lysosome.jpg
Asbestosis and Silicosis
• Sharp particles are endocytosed by macrophages.
• Particles burst lysosomal membranes. Digestive
enzymes spill out
• Another macrophage comes along and the cycle
repeats
• Eventually fibroblasts lay down scar tissue
(collagen) to contain damage. Lung now has a
hard patch which does not function properly –
BLACK LUNG.
• Also, leads to lung cancer.
Experimental techniques for
investigating tracking and
targeting of proteins and vesicle
transport
15_25_methods.jpg
Signal sequence is required
for protein transport to
target organelle
Use of mutants to dissect the protein secretory pathway
15_26_Temp_mutants.jpg
GFP=green fluorescent protein
GFP
protein of interest
1. GFP gene fused to gene coding for protein
of interest
2. Transform cell with GFP-protein gene fusion.
3.Gene is expressed, targeted, protein functions.
4. Localize Green fluorescence with fluorescent.
light microscopy (or confocal)
Control-cytoplasmic GFP, i.e. no protein of interest
fused onto GFP.
• PULSE:Cell is exposed to radioactivelylabelled nucleotides (green) for 3
minutes. The nucleotides are taken up
by the nucleus.
• CHASE:The cell is exposed to an excess
of non-radioactive nucleotides. After
five minutes of chase, some radioactive
molecules are found in the cytoplasm.
• 15 MINUTES:Most of the radioactivity
has moved from the nucleus to the
cytoplasm after 15 minutes of the
chase.
• 30 MINUTES:After 30 minutes, all of
the radioactive label is found outside
the nucleus.
Pulse-chase autoradiography
Pulse chase experiment shows secretory pathway
Radioactive proteins labeled in red
3 min
20 min
120 min
Fig. 8-3 from KarpCell and Molecular
Biology
Time course of protein transport
• Samples are
taken after
various periods
of time and the
location of the
labeled
molecules is
identified.
(CV)
(Z)