Chapter 12: Protein Sorting & Transport

The endoplasmic reticulum is a network of…

membrane enclosed tubes & sacs (cisternae) extending from nuclear membrane throughout cytoplasm

How much of the cell does the ER make up

½ of all cell membranes and 10% of cell volume

What are 3 contiguous membrane domains of the ER and their function

1. Rough ER = protein synthesis

2. Transitional ER = generate vesicles to the Golgi

3. Smooth ER = lipid synthesis

What did Palade do (1975)

revealed the secretory pathway for PM & lysosomal proteins through pulse / chase experiments (followed radiation)

Proteins for secretion or incorporation into ER, Golgi, lysosomes, or PM are initially targeted to…

the ER

In what two ways are polypeptides translocated

1. translational to ER

2. posttranslational

Cytosol → ____ → ____ → ____ → ____

RER → Golgi → Secretory Vesicles → Exterior

What are two ways of protein sorting

1. free ribosomes in cytosol

2. membrane-bound ribosomes

What is cotranslational targeting

ribosome binding to ER mediated by signal sequences within nascent polypeptides

Signal sequences are short…

short (~20aa) N-terminal hydrophobic sequences

Who came up with the signal sequence hypothesis

Sabatini & Blobel

Who supported the signal sequence hypothesis & how

Blobel & Dobberstein; in vitro translation studies using microsomes

5 steps to protein targeting to ER (cotranslational targeting)

1. Signal Recognition Particles (SRPs) bind signal sequence

2. SRP (complex of 6 polypeptides & srpRNA) bind polypeptide / free ribosomes; inhibit translation

3. ER SRP Receptor binds SRP/ribosome complex

4. SRP released, ribosome binds translocon, signal sequence inserted into translocon, translation proceeds

5. Signal sequence cleaved by signal peptidase, releasing polypeptide into ER lumen

Membrane associated proteins are integral proteins…

integral proteins inserted into ER membrane & processed as membrane components

What is common of transmembrane domains

typically a-helical; composed of 20-25 hydrophobic amino acids

What determines membrane associated proteins orientation

determined during translation & translocation

N or C terminal extracellular, multipass

Protein Insertion (I)

1. cytosolic N- or C- terminal

2. signal sequence not cleaved → forms transmembrane domain

3. TM exits translocon

4. orientation depends on signal sequence orientation

Protein Insertion (II)

1. Cytosolic C-terminus = signal sequenced cleaved

2. internal a-helix (stop transfer sequence) forms TM domain

3. remaining sequence translated in cytosol

4. TM domain exits translocon, enters lipid bilayer

Protein Insertion (III)

1. alternating signal sequences & TM stop-transfer sequences

2. alternating sequences: multiple membrane embedded helices connected by loop domains on lumenal & cytosolic sides

ER & Golgi lumens are topologically…

equivalent to cell exterior

polypeptide domains that are transferred to ER lumen are ultimately exposed to what

extracellular space

What are some membrane lipids:

phospholipids, glycolipids, & cholesterol

phospholipids: glycerol…

glycerol derivatives formed on cytosolic side of ER from H2O soluble precursors

Phospholipid synthesis steps

• derived from glycerol derivatives on cytosolic side of ER from H2O soluble precursors

  1. Acetyl groups transferred from CoA to glycerol-3-phosphate

  2. phospholipids inserted into ER membrane

  3. Addition of a polar head groups from phosphatidylcholine, ethanolamine, & inositol

Newly synthesized phospholipids must be transferred to the ____ side of the ER

lumenal

What catalyzes phospholipid translocation

Scramblases - move them around in one side

Golgi Flippases - flip them from side to side

Protein export from the ER occurs via

via vesicles budding from transitional ER

Transport pathway for proteins from ER

ER → Vesicle → ER-Golgi intermediate compartment → Golgi

How is protein sorting controlled

localization signals

• retention KDEL signal

  • Lys - Asp - Glu - Leu

• TM retention (KKXX)

  • Lys - Lys - X - X

Proteins are selectively retrieved from what

ER-Golgi intermediate complex / compartment

What does the Golgi Apparatus do

receive and process proteins for secretion or delivery (endosomes, lysosomes, PM)

What is the structure of the Golgi Apparatus

composed of flattened sacs (cisternae) & associated vesicles

Cis Golgi - Medial Golgi - Trans Golgi

What are the 5 points to Golgi Organization

1. ER-Golgi intermediate compartment (ERGIC) fused with ER vesicles

2. From ERGIC, proteins enter cis Golgi network

3. ER-resident proteins returned

4. Proteins modified & sorted as they move through Golgi stack (medial & trans)

5. Proteins, lipids, & polysaccharides distributed from trans Golgi Network

Constitutuve secretion

protein & lipid delivery to PM continuous, unregulated

regulated secretion

secretion in response to external signals (e.g. hormones)

Intracellular targeting

lysosomal proteins marked by N-terminal (lumenal) or C-terminal (membrane) oligosaccharides

PM is divided into what

Apical & Basolateral domains, each associated with domain specific proteins

Trans Golgi selectively transports proteins to correct…

PM domain (apical or basolateral) (2 types of constitutive secretory vesicles)

Cargo selection, coat proteins, & vesicle budding steps

1. protein sorting

2. membrane budding

3. transport vesicle movement to target

How is transport vesicles formation regulated

by GTP-binding proteins & adaptor proteins (interact with vesicle coat proteins)

Lysosomes

digestive organelles containing ~50 degradative enzymes; mainly hydrolases active @ pH 5

membrane proton pumps actively transport H+ into the ____ interior

lysosome

Early endosomes do what

receive endocytic vesicles from PM

• pass recycled PM molecules to recycling endosomes

• molecules for degradation passed to late endosomes

Late endosome transition is associated with

lowering of pH to ~5.5

Low pH causes release of what from what

hydrolases from Golgi vesicle receptors

receptors then recycled to Golgi