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, 15-16, 15-18, 15-19, 15-21).

Learning ObjectivesI. 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.

pancreas cell

Eukaryotes have many membrane bound compartments

15_02_cell_intestine.jpg

01_24_Organelles.jpg

How are proteins transported intra- and inter-cellularly?

15_05_import_proteins.jpg

15_06_Signal_sequence.jpgSignal sequences direct proteins to specific organelles

Transport of proteins through nuclear pores

• Active transport – requires GTP hydrolysis

• Prospective nuclear proteins have a nuclear localization signal (NLS): -Pro-Lys-Lys-Lys-Arg-Lys-Val-

15_10_unfolded_imprt.jpgProtein transport in mitochondria

Chaperone proteins pull the proteins across the membranes and refold themInside the organelles

15_11_ER.jpg

Structure of ER: (A) Green fluorescent protein fused to ER resident protein; (B) TEM of a thin section

15_12_pool_ribosomes.jpg

Synthesis of cytosolic andER-bound proteins

15_13_ER_signal_SRP.jpgAn ER signal sequence, a signal recognition particle(SRP) and an SRP receptor are required for transportof a protein into the ER

15_14_enters_lumen.jpg

A soluble protein enters the ER lumen via the proteintranslocation channel [Fig. 15-14]

15_15_into_ER_membr.jpg

A single-pass transmembrane protein uses two hydrophobic signal sequences:a N-terminal start transfer sequence and a stop transfer sequence

15_16_double_pass.jpg

A double-pass ER transmembrane protein uses an internal start-transfersequence to integrate into the ER membrane [Fig. 15-16]

A multipass ER transmembrane protein uses many pairs of start and stop sequences

01_25_endocytosis exoc.jpgEndocytosis

Exocytosis(secretion)

Vesicular transport

- Endocytosis - Exocytosis (secretion)

QUIZ: Name the compartments of the secretory and endocytic pathways [Vesicular transport]

3

1

2

4

5

6

Fig. 15-17

lysosome

endosome

Secretory and Endocytic pathways [Fig. 15.24]

15_17_Vesicles_bud.jpg

Different vesicle coats drive budding on different compartments [Table 15-4]

Type of coat Coatproteins

Donor Target

Clathrin Clathrin +adaptin 1

Trans-Golgi Lysosome(viaendosome)

Clathrin Clathrin +adaptin 2

PM endosome

COP-II COPproteins

ER Cis-Golgi

COP-I COPproteins

Golgi Golgi, ER

Table 15-4: Different vesicle coats drive budding on different compartments

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]

15_19_Clathrin_vesicle.jpg

Transport of specific proteins by clathrin-coated vesicles

GTP-binding

15_20_SNAREs.jpg

The specificity of transport vesicles for their target membranes depends onspecific marker proteins, called SNARES

15_21_membr_fusion.jpgSNARE proteins play a central role in membrane

fusion [Fig. 15-21]

1.The vesicle is recognizedby a coiled-coil tetheringprotein and a multisubunittethering complex

2. A RabGTPase bound tothe vesicle stimulates formation of a stable complex of one v-SNAREand 3 t-SNARE helices

3. The v-SNARE/t-SNAREinteraction promotesFusion of membranes

4. Binding of the NSF andSNAPs proteins promotesdissociation of SNAREcomplexes

The SNARE hypothesis for transport vesicle targeting and fusion [Becker]

What kind of chemical modifications does a protein

undergo during vesicular transport?

15_22_glycosylated_ER.jpg

Early glycosylation (N-linked) of proteins in the ER [Fig. 15-22]

15_23_Chaperones.jpgChaperones prevent misfolded or partially assembled Proteins from leaving the ER

[Fig. 15-23]

Cystic fibrosis: a genetic disease in which a plasma membrane transport proteinis slightly misfolded. It would still function if it reached the plasma membrane. Butit 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

Each stack=a dictyosome, consists of cisternae (singular, cisterna)

CIS

TRANS

TGN

cis/trans polarity

Fig. 1-23

Models of Golgi FunctionThere are two competing theories:

Cisternal progression model:New cisternae form continuouslyfrom 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.

In ER, oligosaccharide added, modified.

In Golgi, new sugars added to oligosaccharide

GluManNGln

Protein modifications in Golgi [Fig. 12-6

from Becker]

Predict the location of enzymes, galactosyl transferase andsialic acid transferase

15_28_trans_Golgi_net.jpg

The regulated and constitutive pathways of exocytosis [Fig. 15-28]

Targeting of solublelysosomal enzymesto endosomes andlysosomes by aMannose 6-phosphatetag [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. Phagocytosis

2. Pinocytosis

3. Receptor-mediated endocytosis

4. Intracellular digestion in lysosomes

15_30_white_bloodcell.jpg

Phagocytosis: A white blood cell ingests a bacterium [Fig. 15-30]

15_31_macrophage.jpg

Phagocytosis: A macrophage engulfs two red blood cells [Fig. 15-31]

Autophagic digestion of mitochondria [Becker]

15_32_LDL_enters.jpg

LDL enters cells via receptor-mediated endocytosis [Fig. 15-32]

Receptor mediated endocytosis [Becker Fig. 12-15; ECB Fig. 15-33]

15_34_lysosome.jpg

A lysosome contains hydrolytic enzymes and a H+ pump [Fig. 15-34]

15_35_path_lysosome.jpg

Different pathways leading to intracellular digestion in lysosomes [15-35]

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 requiredfor protein transport to target organelle

15_26_Temp_mutants.jpgUse of mutants to dissect the protein secretory pathway

GFP=green fluorescent protein

GFP protein of interest

1. GFP gene fused to gene coding for protein of interest2. 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 radioactively-labelled 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

Fig. 8-3 from Karp-Cell and Molecular Biology

Pulse chase experiment shows secretory pathway

Radioactive proteins labeled in red

3 min 20 min 120 min

Time course of protein transport• Samples are

taken after various periods of time and the location of the labeled molecules is identified.

(CV)

(Z)