8. Cytoplasmic Transport

<!-- Endomembrane systems -->

Endomembrane system
===================
	* Nuclear Membranes
	* ER - protein synthesis (RER)
	* Golgi - sorting station, protein modification (such as glycosylation)
	* Endosomes
	* Lysosomes

Smooth ER
=========
	* Muscle tissue, kidney tubules, steroid-producing endocrine glands
	* Function: 
		* Synthesis of lipids, stereoid hormones
		* Detoxification - liver P450 cytochrome 
		* Storage of Ca+2 ions
		* IPv3 signals to release

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RER Synthesized Proteins
------------------------
	* **Integral membrane proteins**
	* *Secreted* proteins
	* Souble organelle proteins
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Freely Sythesized Proteins
--------------------------
	* **Peripheral membrane proteins**
	* *Cytoplasmic* proteins
	* *Globular* proteins
	* *Souble* proteins, eg. hemoglobin
	* **Nuclear proteins**
	* **Mitochondria/Chloroplast** proteins

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Transfer
========
	1. Sorting
	2. Budding
	3. Docking
	4. Release

Biosynthetic Pathway
====================
	* Biosynthetic Pathway = Secretory Pathway
		* Constituative
		* Regulated

Endocytic Pathway
=================
	* Endocytic Pathway
		* Uptake exogenous
		* Downreg receptors

Sorting Address 
===============
	* ...

Study of sorting
================
	* Pulse chase -- labeled AA followed by nonlabeled
	* GFP -- transfect into desired cell, can be temp-sensitive
	* Cell free systems -- centrifuge, collect supernatant **microsomes**
	* Yeast are easy to study for mutants
		* Sec 15 -- cannot bud from ER
		* Sec 17 -- vesicles cannot dock w/ golgi
	* RNAi silencers -- block eg. *Mannosidase II*, Golgi resident pro

Signal Sequence
===============
	1. Signal Sequence -- emerges from N-terminus of new protein
	2. **Signal Recognition Particle (SRP)** binds, stopping translation
	3. SRP binds **SRP receptor** at ER
	4. Translation continues through **translocon** (plug removed)
		* A plug normally keeps the translocon shut
	5. Signal Peptidase -- clips off the signal peptide
	* Early conformation, closed conformation, activated conformation, abort... ???

Molecular Chaperones
====================
	* BiP -- provides optimal environment for folding
	* Heat Shock Proteins (HSP) -- Cell is stress, upreg'd to prevent denaturation

Modifications of Proteins
=========================
	* Modifications begin in ER -- co- and post-translationally 
		* Signal peptidase -- removal of signal sequence
		* Chaperones -- folding of protein
		* Addition of oligosacc. co-translationally 
	* Glycosylation 

Glycosylation in ER
===================
	* Flipases -- must be used to transfer sugars into ER from cytosol (clever!)
	* Glycosyltransferases -- transfer oligosac. to proteins
	* Usually Glucose, Mannose, and N-Acetylglucosamine (NAG)

Quality Control
===============
	* Unfolded Protein Response --
		* TODO
	* ER-Associated Degradation (ERAD) --
		* Protein begins with **glu-glu-glu**
		* Glucosidase I -- trims the two terminal glucose residues, leaving one
		* Calnexin -- chaperone recognizes single glucose residue
		* Glucosidase II -- trims the final glucose
		* After several rounds of folding, mannose residues clipped off - destined
		  to be degraded.
		* Ubiquitin -- tags proteins for degradation
		* Proteosome -- degrades ubiquitin-coated proteins

Transport
=========
	* **COP II** coated vesicles - anterograde (forward) transport
		* *ER -> ERGIC -> Golgi (cis -> medial -> trans)*
	* **COP I** coated vesicles - retrograde transport
		* *TGN -> Medial -> Cis -> ERGIC -> ER*
	* **Clathrin coated**
		* *TGN -> PM or Lysozomes/Endosomes*

SAR
===
	* A G-Protein
	* Sec23/34 & Sec13/31 ???
	* When activated, becomes membrane bound
	* Recruits coat proteins, sec proteins, cargo, intrinsic curvature

Docking
=======
	* Coat proteins must fall off
		* Involves Sec23/24 ???
	* GTPgammaS -- non-hydrolyzable analog, makes impossible to fuze w/ acceptor mbr.

COPI Coated Vesicles
====================
	* ARF (vs SAR)


Retrograde transport
====================
	* Retrieval of escaped proteins
	* KDEL sequence -- for ER resident proteins
		* KDEL Receptor - 7tmbr domain - KKXX sequnce recognized
		* Picked up byARF-regulated proteins

Beyond the Golgi
================
	* Regulated secretion -- eg. Neurotransmitter
	* Constitutive secretion -- eg. Albumin
	* Endosomes -> Lysosomes
		* Lysosome-bound proteins have a tag
		* Mannose tag
			* Phosphorylation of mannose occurs at TGN
			* Mannose-6-phosphate
		* Mannose-6-phosphate receptor 
	* Arf recruited
	* GGA adapter -- ??

G-proteins
==========
	* Arf -- In Clathrin-mediated & COPI-mediated
	* SAR -- ???

Transportation Process
======================
	1. Cargo selection
	2. Coating & vesicle budding
	3. Scission
	4. Uncoating (required)
	5. Tethering
		* Rab (gpro) -- ?? 
	6. Docking
		* v-Snares -- on vesicle
		* t-Snares -- on target
		* NSF -- hydrolysis of NSF disassembles SNAREs
	7. Fusion

Endocytic Pathway
=================
	* Bulk phase -- *constituitive, nonspecific*
		* continuous uptake of fluid, eg. pinocytosis
	* Recepter mediated endocytosis (RME) -- requires *specific ligands*
		* often used to internalize/downreg/recycle receptor
		* uptake of essentials - cholesterol, iron

Clathrin-coated pits
====================
	1. Concentration of receptors at *clathrin coated pits*
	2. Coated pits invaginate into cytoplasm, entrapping material
	3. Vesicle formation
	4. Pinch off via **dynamin (g-pro)** "neck"
		* GTP Hydrolysis required, *GTPgammaS will prevent* pinching off
	5. Uncoat -> fuze with lysosome

Clathrin
========
	* **Triskelion** shape
	* AP2 adapter -- links clathrin coat to cargo
		* cf. GGA adapter

Housekeeping Receptors
======================
	* Deliver required material to cell
	* **LDL-R** for cholesterol
	* **Transferrin-R** for iron

Cholesterol metabolism
======================
	* Cholesterol -- lipoprotein for synthesis of hormones, membrane
	* An exogenous/dietary metabolism 
	* LDL bad, HDL good
	

LDL "Bad cholesterol"
=====================
	* Packaging of Cholesterol *since it cannot flow in bloodstream itself*
	* Apolipoprotein B-100 -- wraps around LDL and _binds cell receptor_
	* Experiment: Can change temperature to prevent internalization
	* Cholesterol de-esterified for use by cell


Familial Hypercholestermia (FH)
===============================
	* HMG CoA reductase -- regulates endogenous synthesis of cholesterol
		* Normally influenced by dietary cholesterol
	* Familial Hypercholestermia (FH) -- defect in extracellular binding domain
	* JD mutation -- cytosolic domain affected