Intracellular Compartmentalization

The major intracellular compartments of an animal cell

Compartmentalization of Cells

Membranes
Partition cell Important cellular functions Impermeable to most hydrophobic molecules contain transport proteins to import and export specific molecules Mechanism for importing and incorporating organelle specific proteins that define major organelles

Compartmentalization of Cells
Major Organelles
Nucleus
Cytosol ER Golgi Apparatus Mitochondria and Chloroplast Lysosomes Endosomes Peroxisomes

Relative Volumes Occupied by the Major Intracellular Compartments

INTRACELLULAR COMPARTMENT PERCENTAGE OF TOTAL CELL VOLUME

Cytosol Mitochondria Rough ER cisternae Smooth ER cisternae plus Golgi cisternae Nucleus Peroxisomes Lysosomes Endosomes

54 22 9 6 6 1 1 1

Four distinct families

1) the nucleus and the cytosol, which communicate through nuclear pore complexes and are thus topologically continuous (although functionally distinct); 2) all organelles that function in the secretory and endocytic pathways, including the ER, Golgi apparatus, endosomes, lysosomes, the numerous classes of transport intermediates; 3) the mitochondria;

4) the plastids (in plants only).

Topology governed by evolutionary origins

Organelles arising from pinching off of PM have interior equivalent to exterior of cell

Development of Nucleus and ER

Topology governed by evolutionary origins

Invagination of pm creates organelles such as nucleus that are topologically equivalent to cytosol and communicate via pores

Development of Mitochondria

Endosymbiosis of mitochondria and plastids creates double membrane organelle which have their own genome.

Development of plastids

Compartmentalization of Cells
3 Types of Transport Mechanisms
1. Gated Transport:
gated channels topologically equivalent spaces 2. Transmembrane Transport: protein translocators topologically distinct space 3. Vesicular transport:

membrane enclosed intermediates topologically equivalent spaces

Membrane Trafficking

Membrane Trafficking
Endo-membrane system General concepts of vesicle-mediated traffic Sequence of events beginning with ER and ending at PM Details of Golgi function Golgi to PM pathway The Endocytotic Pathway The Exocytotic Pathway

Endomembrane Network
The endomembrane system is a network of organelles in eukaryotic cells Exocytosis begins in the endoplasmic reticulum The Golgi apparatus modifies and sorts proteins in the exocytic pathway Exocytosis ends at the plasma membrane Endocytosis begins at the plasma membrane Endocytosis ends at the lysosome

The endomembrane system is a network of organelles in eukaryotic cells

The endomembrane system is a set of interconnected organelles that readily exchange materials. The primary functions of the endomembrane system are to control the export (exocytosis) and import (endocytosis) of materials to/from the extracellular space.

The endomembrane system is a network of organelles in eukaryotic cells

Membrane-bound compartments called vesicles shuttle between organelles in the endo-membrane system and are responsible for carrying material from one organelle to another.
The creation, transport, targeting, and fusion of vesicles occurs in nine steps.

The endomembrane system controls molecular transport in/out of cell

Endocytic and exocytic pathways Endomembrane system

The endocytic and exocytic pathways.

Getting molecules into cells: crossing the plasma membrane

Diffusion across the plasma membrane water, gases, small molecules Protein-mediated transport ion channels, transporters
O2

extracellular
Ca2

Y Y

Pore formation toxins

Membrane fusion viruses Formation and internalization of membrane-limited vesicles Endocytosis

O2

Ca2

cytoplasm

Membrane Trafficking: Vesicular Transport

Transport large particles or fluid droplets through membrane in vesicles uses ATP Large packets of substances and engulfed cells move across the plasma membrane by processes of endocytosis and exocytosis Exocytosis transport out of cell Endocytosis transport into cell phagocytosis engulfing large particles pinocytosis taking in fluid droplets receptor mediated endocytosis taking in specific molecules bound to receptors

Membrane lipids and proteins move to and from the plasma membrane during these processes

Vesicular Transport
RER to cis Golgi Modified in Golgi (glycosylation, phosphorylation) Sorted at trans Golgi network into

Lysosomal (endocytosis)
Regulated (exocytosis) constitutive (exocytosis)

Vesicles shuttle material between organelles in the endomembrane system

Donor/acceptor compartments Vesicle-mediated transport Exocytosis, endocytosis, and fusion of vesicles

In vesicle-mediated transport, a membrane-bound vesicle buds from one compartment and fuses with another.

The Golgi apparatus plays a central role in vesicular traffic within cells.

The Golgi apparatus modifies and sorts proteins in the exocytic pathway
The Golgi apparatus is organized into discrete compartments called cisternae. The cisternae are stacked on top of one another, and are classified as cis, medial, or trans according to their relative location within the overall Golgi structure. Golgi-resident proteins are primarily responsible for modifying proteins undergoing exocytosis. They are retained in the Golgi apparatus by transmembrane Golgi retention sequences.

The Golgi apparatus modifies and sorts proteins in the exocytic pathway
The extreme ends of the Golgi apparatus are elaborated into long, tubular structures called the cis Golgi network and trans Golgi network. Both Golgi networks sort proteins into vesicles targeted to different locations. The trans Golgi network is especially effective at sorting a large number of proteins into many distinct vesicle types.

Proteins exiting the ER join the Golgi apparatus at the cis Golgi network. The Golgi apparatus consists of a collection of stacked compartments.

Pathway Through Golgi Apparatus

1. Molecules come in vesicles
2. Vesicles fuse with Golgi membrane 3. Molecules may be modified by Golgi 4. Molecules pinched-off in separate vesicle 5. Vesicle leaves Golgi apparatus 6. Vesicles may combine with plasma membrane to secrete contents

The Golgi Apparatus has two major functions:

1. Modifies the N-linked oligosaccharides and adds Olinked oligosaccharides.
2. Sorts proteins so that when they exit the Trans Golgi Network (TGN), they are delivered to the correct destination.

Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments.

1. Sorting in TGN 2. Protection from protease digestion 3. Cell to cell adhesion via selectins

Once proteins that dont normally reside in the ER are properly folded, they are transported to the golgi apparatus.

Three main types of coated vesicles

Each type of vesicle is named according to its primary coat proteins: - Clathrin vesicles transport proteins from plasma membrane or the trans-Golgi network to late endosome. - COPI vesicles transport proteins in between Golgi cisternae and from the cis-Golgi back to the rough ER. - COPII vesicles transport proteins from the rough ER to the Golgi.

The three main types of coats involved in vesicle-mediated transport :

Model for the formation of a clathrin-coated pit and the selective incorporation of integral membrane proteins into clathrin-coated vesicles

COPII coated vesicles transport via the vesicular tubular cluster (vtc) to the cis-Golgi network.

The protein coating is removed and the vesicles fuse with each other to form the vtc. The vtc is motored by kinesin (motor protein) along microtubules that function like engine on rail tracks. The vtc fuses with the cis-Golgi network. COPI transports vesicles back to ER

It is uncertain how proteins move through the Golgi apparatus.

Stationary compartments with vesicles transporting between compartments.

Large moving compartments that mature into the TGN, and return enzymes to trailing compartments by retrieval vesicles.

The trans Golgi network (TGN) sorts proteins exiting the Golgi apparatus

Dynamin uses GTP to regulate scission of a vesicle from a donor compartment.

TGN sorting mechanisms are complex

Many different cargo selection mechanisms
PTM (post-translational modifications) Protein aggregation Signal receptor Lipid raft

Budding of vesicles at the TGN likely occurs by several different mechanisms

3 different mechanisms have been proposed Curvature-inducing proteins Modification of membrane phospholipids Phospholipid asymmetry

Vesicle-mediated transport - 9 steps

1) 2) 3) 4) 5) 6) 7) 8) 9) Cargo selection Budding Scission Uncoating Transport Tethering Docking Fusion Disassembly

Vesicle-mediated transport occurs in 9 steps.

Endocytosis and Exocytosis

Endocytosis: a process of uptake of extracellular material by engulfing it within cell, including receptor-mediated endocytosis, phagocytosis and pinocytosis.

Exocytosis: a process of release of intracellular molecules (such as hormones, secretory proteins) contained within a membranebounded vesicle by fusion of the vesicle with its plasma membrane.

Endocytosis
Small region of the plasma membrane invaginates to form membrane-limited vesicles. Internalized molecules retain topology (lumen = extracellular) Cargo can be specifically selected (receptor) Destination of cargo can be controlled;

Some of the functions of endocytosis

Nutrient uptake Plasma membrane protein regulation and/or degradation Synaptic vesicle recycling Trans-cellular signaling

Exploitation: virus and toxin entry into cells

Three Pathways of Endocytosis

Phagocytosis
Pseudopods engulf target particle and merge as a vesicle, which fuses with a lysosome in the cell

Pinocytosis
Extracellular fluid is captured in a vesicle and brought into the cell;

Receptor-mediated endocytosis
Specific molecules bind to surface receptors, which are then enclosed in an endocytic vesicle

Phagocytosis: engulfment of particles

Ingestion of microbes or particles by a cell Transports specific substance Relatively large in size (~.75micro m)

Phagocytosis in Multicellular organisms

occurs only in specialized cells like macrophages, dendritic cells and neutrophils. capture and destroy pathogens and particulate antigens essential component of the immune response Fc- and complement-receptor mediated phagocytosis named for binding specificity for antibody tail region called Fc (Fragment, crystallizable)

Phagocytosis in Macrophage: The cells receptors in the plasma membrane enable them to recognize their targets. For example, macrophages have receptor that recognizes phosphatidylserine which becomes exposed on the surface of dead cells.

Pinocytosis: internalization of fluid

Generated at sites of ruffling at the plasma membrane Non-specific in substance it transports

Classified as macropinocytosis (vesicles > 1 mm in diameter) and micropinocytosis (vesicles < 200 nm in diameter)

Receptor - Mediated Endocytosis :

A selective process Involves formation of vesicles at surface of membrane Vesicles contain receptors on their membrane Receptors bound to specific target molecule Clathrin-coated vesicle in cytoplasm uptake of LDL from bloodstream If receptors are lacking, LDLs accumulate and hypercholesterolemia develops

Receptor-Mediated Endocytosis
Nutrient Uptake (LDL,transferrin, etc.) Membrane Recycling Membrane Protein Recycling Antigen Uptake Synaptic Vesicle Recycling Signaling Receptor Down-Regulation

Receptor Mediated Endocytosis

Receptor-Adaptin Association Nucleates Pit Formation

Receptors Bind Cargo. Clathrin Adaptins [AP1(Golgi) or AP2(PM)] bind to Receptor Tail Sequences. Coated Pits Form and Pinch Off into Coated Vesicles

GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles

Dynamin-GTP forms a collar around the neck of a coated Pit. GTP hydrolysis by dynamin is required for pinching off.

Endocytosis begins at the plasma membrane

The onset of endocytosis is most often indicated by the clustering of cargo receptors on the plasma membrane, accompanied by the assembly of a clathrin coat on the cytosolic face of the cluster. In micrographs, this structure resembles a pit in the membrane, so it is often called a coated pit. Coated pits complete the nine steps of vesicle transport and deliver the vesicle to an organelle called the endosome.

Clathrin stabilizes the formation of vesicles

The role of clathrin in endocytosis.

Caveolin mediated endocytosis

Caveolae are 50-100 nm invaginations on the cells surface
Caveolin, a membrane protein, is the coat protein of caveolae

Undergo endocytosis in response to a signal (ex. SV40 binding) in a cholesterol- and dynamin-dependent fashion Internalized caveolae recruit actin to form comet tails Upon internalization caveolae are delivered to novel endosomal compartments known as caveosomes

The endosome sorts proteins in the endocytic pathway

The endosome is formed by the fusion of endocytic vesicles with specific vesicles that bud from the TGN. The endosome sorts materials arriving from the plasma membrane; cargo receptors are recycled to the plasma membrane, while cargo remains in the endosome. The lumen of the endosome is slightly acidic relative to the extracellular space, and this acidity is key to the sorting mechanism. This sorting mechanism is very different from the sorting mechanisms used in the Golgi apparatus.

The endosome is subdivided into early and late compartments

Specific interacting molecules ensure correct vesicles fuse with vesicles from TGN or early endosomes Proton pump proteins play central role in sorting/activation of endosomal contents

Maturation of endosomes to form lysosomes.

The endocytic pathway is divided into the early endosomes and late endosomes pathway.
Materials in the early endosomes are sorted:
Integral membrane proteins are shipped back to the membrane; Other dissolved materials and bound ligands retained. Dissociation of internalized ligand-receptor complexs in the late endosomes: Molecules that reach the late endosomes are moved to lysosomes.

Endocytosis ends at the lysosome

Complete degradation of endocytosed materials takes place in the lysosome. The lysosome is likely generated from the endosome in several ways, and requires fusing a vesicle from the TGN that contains essential proton pump proteins and digestive enzymes with the endosome. Once digested, the cargo building blocks (sugars, nucleosides, amino acids, etc.) are transported into the cytosol for reuse.

Lysosomes: membranous organelles filled with

digestive enzymes
Breakdown endocytosed materials
Through phagocytosis or receptor mediated endocytosis

Breakdown old organelles (residual body)

Autophagy by autophagosome

One ultimate destination of some proteins that arrive in the TGN is the lysosome. These proteins include acid hydrolases.

Lysosomes are like the stomach of the cell. They are organelles surrounded by a single membrane and filled with enzymes called acid hydrolases that digest (degrade) a variety of macromolecules. A vacuolar H+ ATPase pumps protons into the lysosome causing the pH to be ~5.

Digestion of cargo molecules in the lysosome.

The macromolecules that are degraded in the lysosome arrive by endocytosis phagocytosis or autophagy.

Phagocytosis vs. Autophagy

Phagocytosis

lysosomes

Autophagy

pH is used in 3 ways to control endocytosis

1) the acidic environment in endosomes helps sort cargo from receptors 2) the relatively neutral pH of the ER and Golgi apparatus keeps the hydrolytic enzymes from digesting these organelles 3) the enzymes requirement for a strong acid environment protects the endomembrane system from digesting itself

Vesicular Transport: Exocytosis

Secreting material or replacement of plasma membrane

Exocytosis
Vesicle moves to cell surface Membrane of vesicle fuses Materials expelled orCell discharges material Reverse of endocytosis
Where do newly synthesized membrane and secretory proteins need to go and how do they get there?
Secretion (constitutive and regulated) PM protein delivery (polarized and non-polarized cells) Lysosomal targeting

Constitutive (un-regulated) and regulated secretion

Constitutive secretion/ exocytosis of plasma membrane proteins

Delivered via membrane vesicles directly from the TGN to the cell surface Share same vesicles as constitutively secreted proteins Remarkably little is known about how plasma membrane proteins are sorted into secretory vesicles May be more than one class of carrier vesicles
A model protein for studying the secretory pathway (shown here tagged with GFP)

Regulated secretion
Occurs in endocrine, exocrine and neuronal cells
Insulin secretion in pancreatic b-cells Trypsinogen secretion in pancreatic acinar cells

Exocytosis occurs in response to a trigger (ex. Ca2+)

2 mechanisms for controlling the final steps of exocytosis

Constitutive secretion constant Regulated secretion Controlled by signaling proteins Secretory vesicles (zymogen granules) Condensing vacuole

Figure 09.11: Transmission electron micrograph of clathrin-coated pits and vesicles at the oocyte surface.

Exocytosis ends at the plasma membrane

Cells regulate the last stage of exocytosis (fusion) for most exocytic vesicles, to control when and how much material is released into the extracellular space and to control the delivery of membrane-associated proteins to the plasma membrane. Controlled secretion is also called regulated secretion, and is under the control of signaling pathways.

Transcytosis : provides a way to deliver proteins across epithelium.

Transport of antibodies in milk across the gut epithelium of baby rats.

Acidic pH of the gut favor association of antibody with Fc receptor whereas the neutral pH of the extracellular fluid favors dissociation.

Transcytosis: a closer look

lumen

Transcytosis: transport of macromolecular cargo from one side of the cell to the other Transcytosis is also utilized in the biosynthetic trafficking of some PM proteins pIgA-receptor is a model for studying transcytosis
Contains sorting information in its cytoplasmic tail pIgA is secreted into the the gut lumen, bile and milk as part of the mucosal immune response

pIgA-R

pIgA

Blood/interstitial

synthesizes IgA Tuma and Hubbard (2003)