chapter 15 intracellular compartments and transport essential cell biology third edition copyright...

Chapter 15Intracellular Compartments and

Transport

EssentialCell Biology

Third Edition

Copyright © Garland Science 2010Hilary [email protected]

What we see in most textbooks:

and what it really looks like:

Figure 15-1 Essential Cell Biology (© Garland Science 2010)

Table 15-2 Essential Cell Biology (© Garland Science 2010)

The real numbers

Overview

• Briefly describe membrane-enclosed organelles of eukaryotic cells and their functions

• Discuss how the protein composition of each organelle/compartment is formed and maintained

• Discuss how organelles communicate with each other• Vesicles

Membrane-Enclosed Organelles


Internal Membranes Create Enclosed Compartments and Organelles - Segregating

Metabolic Processes

Examples:

- Separate glycolysis from glycogenesis

- Separate synthesis of protein bonds from hydrolysis of protein bonds


Basic set of Organelles Found in Most Animal Cells


Functions of Organelles


Evolution of the ER and Nuclear Membranes

Single compartmentPlasma membrane invaginated forming a two-layered envelope of membrane surrounding the DNA

Endomembrane System

Intracellular membranes may have evolved from invagination of the plasma membrane

Protein Sorting

• Cell must duplicate its membrane-enclosed organelles before dividing

• Most organelles formed from preexisting organelles then divide and are distributed between daughter cells

• Non-dividing cells continuously generate proteins and replace proteins that have been degraded

• Proteins need to be sorted correctly – organelle membrane proteins, organelle lumen

proteins, secreted proteins – HOW? 3 Mechanisms

How are proteins sorted into discrete locations?

1. Cytosol into the Nucleus

– Nuclear pores - penetrate the inner and outer membranes

– Selective gates– Transport specific

molecules– Allow passive diffusion of

smaller molecules


How can proteins cross a phospholipid bilayer? 1. Nuclear Pores

2. Cytosol into ER, mitochondria, chloroplast

– Transported across membrane by protein translocators - located in the membrane

– Protein usually has to unfold to snake through the membrane

– Similar to bacteria


How can proteins cross a phospholipid bilayer? 2. Protein Translocators

3. ER onward and from one compartment of endomembrane system to another

– Transport vesicles - loaded with cargo of proteins from the interior space of one compartment

– Discharge cargo into second compartment by fusing with the membrane

– Membrane components also delivered (lipids and proteins)


How can proteins cross a phospholipid bilayer? 3. Transport Vesicles


Signal Sequences - direct proteins to correct organelleLocalization sequences

Conserved AA sequence that acts as a molecular “address” telling the cell where this protein needs to live in the cell!


If localization signals are removed, the protein does not arrive at the required destination

How do Proteins Enter the Nucleus?


Nuclear envelope - defines nuclear compartment - formed from two concentric membranes

Inner nuclear membrane - contain proteins that act as binding sites for the chromosomes and provide anchorage for the nuclear lamina

Nuclear lamina - protein filaments that provide structural support for the nuclear envelope

Outer nuclear membrane - membrane similar composition as the ER membrane (continuous with)Nuclear pores - form the gates which all molecules enter or leave the nucleus

Architecture of the nucleus

Figure 15-8a Essential Cell Biology (© Garland Science 2010)

Nuclear pores contain ~30 different proteinsH2O filled passages - small water-soluble molecules can pass freely between nucleus and cytosolJumble meshwork of proteins inhibit larger molecules from passing through the poreNuclear Localization Sequence (NLS) Larger molecules need an NLS to pass through the pore - 1 or 2 short sequences of positively charged lysines or arginines

Nuclear Pore Complex – A Gate

Figure 15-8b Essential Cell Biology (© Garland Science 2010)

EM - side view of two nuclear pore complexes

EM - face-on view of nuclear pore complexes

EM of Nuclear Pores


NTR - grab onto sequences within the tangle of nuclear pore to carry the cargo into the nucleus

Nuclear transport receptors actively transport proteins through nuclear pores


Importing Proteins into the Nucleus Requires Energy - GTP hydrolysis

Similar process used to carry mRNA out of the nucleus in the cytoplasm

Proteins remain in fully folded conformation! Different than transport mechanisms into other parts of the cell.

© Sarah E Golding PhD.

How do Proteins Enter the Mitochondria and Chloroplasts?


Proteins that enter have an N-terminal signal sequence (red)Proteins translocate across both membranes simultaneously at specific sitesProteins are unfolded as they are transportedSignal sequence removed after translocation completed

Proteins unfold in order to enter Mitochondria and Chloroplasts

HELPED BY CHAPERONES TO TRANSFER AND RE-FOLD!

How do Proteins Enter the Endoplasmic Reticulum?

• Entry point for proteins destined for other organelles as well as the ER itself

• Proteins destined for the Golgi apparatus, lysosomes, endosomes, cell surface all first enter the ER from the cytosol

• Once in ER proteins do not return to the cytosol but rather travel via vesicles

© Sarah E Golding PhD.Figure 15-12 Essential Cell Biology (© Garland Science 2010)

ER - most extensive membrane system in a eukaryotic cell

1. Water-soluble proteins - translocated across ER membrane into the ER lumen

• Destined for secretion or for the lumen of an organelle

2. Prospective transmembrane proteins - partially translocated across ER membrane and become embedded in it

• Destined to stay in ER membrane, membrane of another organelle, or plasma membrane

• Directed to ER by ER signal sequence - 8 or more hydrophobic amino acids

2 kinds of proteins transferred from the cytosol to the ER


Proteins that enter the ER

begin to enter the ER

membrane before the

polypeptide chain has been

completely synthesized


ER signal sequence guided to the ER membrane by: 1. A signal-recognition particle (SRP) - in the cytosol binds to the ER signal sequence when it is exposed on the ribosome 2. An SRP receptor - embedded in membrane of the ER - recognizes the SRP

ER localization signals are recognized by SRPs


• ER signal sequence - usually N-terminus - functions to

open channel• Remains bound to channel as remainder of

protein chain threaded through membrane as a large loop

• ER signal cleaved once proteins have crossed!

Soluble proteins cross ER to enter lumen

How are Transmembrane Proteins Transported into the Membrane?


Single-pass Transmembrane Proteins

ER signal cleaved once proteins have crossed!

Double-pass Transmembrane Protein

Start transfer-sequence - internal signal sequence used to start the protein transfer - never removed from peptide!


Need additional pairs of stop and start sequences

– One sequence reinitiates translocation further down peptide chain

– The other stops translocation and causes polypeptide release and so on

– Stitched into membrane as they are synthesized

Multi-pass Transmembrane Proteins

Vesicular Transport

http://www.youtube.com/watch?v=rvfvRgk0MfA

http://www.youtube.com/watch?v=rvfvRgk0MfA

• Two types – secretory pathway and endocytic pathway

• Secretory pathway– Entry into the ER is the first step - pathway to

another destination– Initial destination is the Golgi apparatus– From Golgi to other compartments - carried out by

budding and fusion of transport vesicles– Extend outward: ER plasma membrane

• Endocytic pathway– Extend inward: plasma membrane lysosomes– COMMUNICATION between cells!

Vesicular Transport


Secretory pathway - RED arrowsEndocytic pathway - GREEN arrows

Secretory and Endocytic Pathways

• Vesicles that bud from membranes have a distinctive protein coat on cytosolic surface - coated vesicles

• After budding from parent organelle - sheds the coat allowing the vesicle to interact directly with the membrane it will fuse to

• Cells produce different types of coated vesicles – focus on Clathrin• Two functions of the coat:

1. Shapes the membrane into a bud2. Helps capture molecules for onward transport

Vesicle Budding – Assembly of a Protein Coat

Figure 15-19b Essential Cell Biology (© Garland Science 2010)

Clathrin coated vesicles

http://www.youtube.com/watch?v=eRslV6lrVxY



Figure 15-19a Essential Cell Biology (© Garland Science 2010)

• Clathrin - best studied vesicles have coats made largely of clathrin • Bud from the Golgi apparatus on the outward secretory pathway• Bud from the plasma membrane on the inward endocytic pathway

• Electron micrograph (EM) showing a clathrin-coated vesicle forming

• Assemble into a basketlike network on the cytosolic surface of the membrane • starts shaping the membrane into a vesicle

Clathrin forms a “cage” to carry vesicles to the membrane


Adaptins - secure clathrin coat to vesicle membrane and help select the cargo moleculesDynamin - small GTP binding protein - assembles around neck of invaginated coated pit

Causes ring to contrict - pinching off vesicle from membrane

Hydrolysis of GTP and help of other proteins to pinch off vesicle

Clathrin coated vesicles transport select cargo molecules


Different adaptins - adaptins that bind cargo receptors in the plasma membrane not the same as those that bind cargo receptors in the Golgi apparatus

-Reflects differences in cargo molecules from each source

Adaptin proteins are specific to destination

• Actively transported by motor proteins that move along the cytoskeleton

• Learn More about this in Chapter 17

How are vesicles transported through the cytosol?

http://www.biozentrum.unibas.ch/research/groups-platforms/overview/unit/schoenenberger/


Rab proteins - surface of the vesicle are recognized by tethering proteins on the cytosolic surface of the target membrane Specific combination of Rab proteins and tethering proteins - identify membrane type Ensure vesicles fuse only with the correct membraneSNAREs - transmembrane proteins important for

docking the vesicle in place SNAREs on the vesicle (v-SNAREs) interact with complementary SNAREs on the target membrane (t-SNAREs)

Vesicle Fusion - deliver its cargo and adds vesicle membrane to organelle

Vesicle Reaches Target - Recognize and Dock with the Organelle


• Fusion requires the two lipid bilayers come within 1.5nm of each other so lipid can intermix

• Need to displace water from hydrophilic surface of the membrane

• After pairing the v-SNAREs and t-SNAREs wrap around each other = pulls two membranes into close proximity

Membrane fusion sometimes needs a specific molecular signal

Intracellular bacteria

• Chlamydia spp.• Mycobacterium• Salmonella

• WHY? Reduced genome sizes compared to extracellular bacteria• E. coli = 4.6 megabases• Chlamydia = 1.3 megabases

• Do not have the genes to synthesize many essential nutrients – e.g. Amino acids

• Must parasitize these from their host!• Take advantage of vesicular trafficking

and hijack nutrient-rich vesicles!

Many intracellular bacteria target Rab Proteins!

• Mycobacterium tuberculosis acquires iron and other nutrients by targeting recycling endosome and trans-Golgi Rab Proteins

• Chlamydia spp. acquire sphingolipids and amino acids by targeting trans-Golgi Rab Proteins

Uninfected human epithelium cells (left) with compact Golgi band close to the cell nucleus (blue) and cells infected with Chlamydiae trachomatis with Golgi fragments (red) which accumulate around the bacterial inclusion (green).

http://www.rki.de/EN/Content/Institute/DepartmentsUnits/JuniorGroups/JRG5.html

How do proteins cross the plasma membrane? Secretory Pathways

• Exocytosis - newly made proteins, lipids, and carbohydrates are delivered from the ER, via the Golgi apparatus, to the cell surface by transport vesicles that fuse with the plasma membrane

• FIXED sequence of membrane-enclosed compartments - often chemically modified en route

Exocytosis

• Disulfide bonds are formed between cysteine side chains

Remember? Stabilize protein structure• Glycosylation - covalent attachment of

short oligosaccharide side chains Remember? Protect extracellular proteins,

serve as transport signal form carbohydrate layer, help with cell to cell recognition

– Carried out by glycosylating enzymes in ER but not in the cytosol

Chemical modifications which occur in the ER


• Glycosylation - addition of oligosaccharide side chains at asparagine residues, residues added en bloc

• 14-sugar oligosaccharide is originally attached to specialized lipid in ER membrane – dolichol

• Not all proteins in ER are glycosylated. Requires specific tripeptide sequence adjacent to the modified asparagine.

Glycosylation

This is the beginning of protein modification! Proteins are further modified in the Golgi.

Proteins that function in ER - have ER retention signalExit from ER highly selective

Proteins that fold incorrectly or multi-meric proteins that fail to assemble properly are retained in ER and bind to chaperone proteins

Chaperone proteins holds proteins in ER until proper folding occurs - if does not occur proteins are degraded

Regulated ER Exit – protein quality control!



Unfolded protein response (UPR)- when cells protein production exceeds carrying and folding capacity

UPR prompts the cell to make more ER - including all molecular

machinery (lots of transcription!)

UPR allows cell to adjust size of ER to met the cellular needs

- if misfolded proteins continue to accumulate (out of control!)- UPR can direct cell to undergo apoptosis

Unfolded protein response (UPR)

• Oligosaccharides added in ER are further modified – removing or adding sugars

Golgi apparatus• Usually located near cell nucleus• Collection of flattened membrane-enclosed

sacs called cisternae with two distinct faces:– Cis face is adjacent to the ER– Trans face points toward the plasma membrane

• Outermost cisterna on each face is connected to a network of tubules and transport vesicles

Proteins further modified and sorted in the Golgi apparatus

The Golgi apparatus! Molecular post office

Proteins enter at Cis-face: move through and exit Golgi or return to ER

Proteins exit at trans-face: transported to Plasma membrane or lysosomes



Secretory proteins are released by exocytosis


Secretory cells - Regulated and Constitutive (operates continually) Pathways

Constitutive secretion – all Eukaryotic cells NO Signal Sequence• Proteins incorporated into plasma membrane, extracellular matrix or are

signaling molecules Regulated secretion – specialized cells need signal to stimulate fusion with the plasma membrane and release cargo to cell exterior

• Example: Insulin!

Constitutive Secretion vs Regulated Secretion


Example: Release of insulin from a secretory vesicle of a pancreatic cell Signaled to release by an increase in glucose levels in the blood

Insulin is released by regulated secretion

How do proteins enter the cell? Endocytic Pathways

Review video: http://www.youtube.com/watch?v=SGBiy1HlWH8

• Endocytosis - eukaryotic cells continuously take up fluid, as well as large and small molecules

• Material to be ingested enclosed by a small portion of plasma membrane - buds inward and then pinches off to form an intracellular endocytic vesicle

Endocytosis

• Pinocytosis (“cellular drinking”) - ingestion of fluid and molecules via small vesicles (< 150nm in diameter)– Carried out predominantly by clathrin-

coated vesicles

• Phagocytosis (“cellular eating”) - ingestion of large particles via large vesicles called phagosomes (generally > 250nm in diameter)– Only “phagocytic” cells do this

2 main types of Endocytosis


Macrophage engulfing a pair of red blood cells

Phagocytic cells can ingest whole cells!

Pinocytosis is indiscriminate

Receptor-mediated Endocytosis = controlled pinocytosis

– More efficient - macromolecules bind to complementary receptors on the cell surface and enter the cell as receptor-macromolecule complexes in clathrin-coated vesicles

– Selective concentrating mechanism– Increases efficiency of internalization of

particular macromolecules more than 1000-fold compared to pinocytosis

– Example: Cholesterol

Receptor-mediated Endocytosis


LDL enters cells by receptor-mediated endocytosis

• Two sets of endosomes– Early endosomes: Just beneath plasma

membrane - mature into late endosomes as they fuse with each other

– Late endosomes: Closer to the nucleus• Interior of endosome kept acidic by proton

pump in the endosomal membrane– pH 5-6– Low pH causes receptors to release their cargo

• Main sorting station in the inward endocytic pathway– trans-Golgi is the sorting station for exocytic or

secretory pathway!

Endosomes – site for sorting of imported macromolecules

Routes taken by receptor once they enter the endosome differ according to the receptor type

1. Recycling

2. Lysosomes

3. Transcytosis



Lysosome contains hydrolytic enzymes and a proton pump

Digest worn out organelles and extracellular materials

pH ~5 (acidic)

Unique membrane - contains transporters to allow products of the digestion to be transferred to the cytosol to be either excreted from cell or used by the cell

Lysosomes are the Principal Sites of Intracellular Digestion

Acidic pH and destructive enzymes are contained within membrane!


The lysosome - The cell dumping station!

Endocytosed, phagocytosed and autophagosomal material are trafficked to the lysosome for destruction!

Autophagy is the destruction of an organelle – envelopes organelle and takes it to the lysosome

chapter 15 intracellular compartments and transport essential cell biology third edition copyright...

Documents

proteins figure

nucleus slide

plasma membrane slide

organelle membrane proteins

nuclear compartment

gate slide

protein sorting slide

nucleus nuclear pores