Intracellular Compartments and Protein Sorting

Intracellular Compartments

► A eukaryotic cell is elaborately subdivided into functionally distinct, membrane-enclosed compartments.

► Each compartment, or organelle, contains its own characteristic set of enzymes and other specialized molecules, and complex distribution systems transport specific products from one compartment to another.

► Proteins confer upon each compartment its characteristic structural and functional properties.

► They catalyze the reactions that occur in each organelle and selectively transport small molecules into and out of its interior, or lumen.

► Proteins also serve as organelle-specific surface markers that direct new deliveries of proteins and lipids to the appropriate organelle.

► An animal cell contains about 10 billion (1010) protein molecules of perhaps 10,000 - 20,000 kinds, and the synthesis of almost all of them begins in the cytosol.

► Each newly synthesized protein is then delivered specifically to the cell compartment that requires it.

Intracellular Compartments and Protein Sorting

►Functionally distinct membrane bound organelles

►10 billion proteins of 10,000-20,000 different kinds

►Complex delivery system

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


All Eukaryotic Cells Have Same Basic Set of Membrane Bound Organelles

The Neucleus and Cytoplasm ► The nucleus contains the main genome and is the principal site of DNA

and RNA synthesis.

► The surrounding cytoplasm consists of the cytosol and the cytoplasmic organelles suspended in it.

► The cytosol, constituting a little more than half the total volume of the cell, is the site of protein synthesis and degradation.

► It also performs most of the cell's intermediary metabolism that is, the many reactions by which some small molecules are degraded and others are synthesized to provide the building blocks for macromolecules

► About half the total area of membrane in a eukaryotic cell encloses the labyrinthine spaces of the endoplasmic reticulum (ER).

► The ER has many ribosomes bound to its cytosolic surface; these are engaged in the synthesis of both soluble and integral membrane proteins, most of which are destined either for secretion to the cell exterior or for other organelles.

► Whereas proteins are translocated into other organelles only after their synthesis is complete, they are translocated into the ER as they are synthesized.

► This is why the ER membrane is unique in having ribosomes tethered to it.

► The ER also produces most of the lipid for the rest of the cell and functions as a store for Ca2+ ions.

► The ER sends many of its proteins and lipids to the Golgi apparatus.

Endoplasmic Reticulum

Golgi Complex

Vesicle budding from rough endoplasmic reticulum

Fusion of vesicle with Golgi apparatus

Migrating transport vesicle

Protein

Proteins

Transport vesicle

Golgi apparatus

Cisternae

Ribosome

trans face

cis face

► The Golgi apparatus consists of organized stacks of disclike compartments called Golgi cisternae; it receives lipids and proteins from the ER and dispatches them to a variety of destinations, usually covalently modifying them en route.

► Mitochondria and (in plants) chloroplasts generate most of the ATP used by cells to drive reactions that require an input of free energy; chloroplasts are a specialized version of plastids, which can also have other functions in plant cells, such as the storage of food or pigment molecules.

► Lysosomes contain digestive enzymes that degrade defunct intracellular organelles, as well as macromolecules and particles taken in from outside the cell by endocytosis.

► On their way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes.

► Peroxisomes are small vesicular compartments that contain enzymes utilized in a variety of oxidative reactions.

Mitochondria Chloroplast

Cellular Compartments

► In general, each membrane-enclosed organelle performs the same set of basic functions in all cell types.

► But to serve the specialized functions of cells, these organelles will vary in abundance and can have additional properties that differ from cell type to cell type.


Major Organelles

►Nucleus

►Cytosol

►ER

►Golgi Apparatus

►Mitochondria and Chloroplast

►Lysosomes

►Endosomes

►Peroxisomes


►Occupy 50% cell volume

►Perform same basic function

►Vary in size and abundance

►May take on additional functions

►Position dictated by cytoskeleton

Protein Sorting

► All proteins begin being synthesized on ribosomes in the cytosol, except for the few that are synthesized on the ribosomes of mitochondria and plastids.

► Their subsequent fate depends on their amino acid sequence, which can contain sorting signals that direct their delivery to locations outside the cytosol.

► Most proteins do not have a sorting signal and consequently remain in the cytosol as permanent residents.

► Many others, however, have specific sorting signals that direct their transport from the cytosol into the nucleus, the ER, mitochondria, plastids, or peroxisomes; sorting signals can also direct the transport of proteins from the ER to other destinations in the cell.

Types of Transport Mechanisms

1. Gated Transport:

the protein traffic between the cytosol and nucleus

gated channels

topologically equivalent spaces

2. Transmembrane Transport:

protein translocators

topologically distinct space

unfolded protein

Cytosol to ER

3. Vesicular transport:

membrane enclosed intermediates

The transfer of soluble proteins from the ER to the Golgi apparatus

topologically equivalent spaces

Compartmentaliztion of Cells

Families of Intracellular Compartments: 1. nucleus and cytosol 2. organelles in the secretory pathway 3. mitochondria 4. plastid

Transport guided by: 1. sorting signals in transported proteins 2. complementary receptor proteins

1. Signal Sequence

continuous sequence of 15-60 aa

sometimes removed from finished protein

sometimes a part of finished protein

2. Signal Patch

specific 3d arrangement of atoms on protein surface; aa’s distant

persist in finished protein

Types of Sorting Signals in Proteins

The Endoplasmic Reticulum & Protein Trafficking

ER and Protein Trafficking

Endoplasmic Reticulum ► Occupies >= 50% of cell volume

► Continuous with nuclear membrane

► Sequestration of Ca++ from the cytoplasm.

► Central to biosynthesis of macromolecules used to construct other organelles

► Trafficking of proteins to ER lumen, Gogli, lysosome or those to be secreted from cell

Abundant smooth ER

in steroid-hormone-

secreting cell

A 3-D reconstruction

of ER in liver cell

Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)

ER and Protein Trafficking ER removes 2 types of proteins from cytosol:

1. Transmembrane proteins, partly translocated across ER, embedded in it

Some proteins function in the ER, but many are destined to reside in the plasma membrane or the membrane of another organelle.

2. Water - soluble proteins, translocated into lumen

Either for the lumen of an organelle or for secretion.

Figure 12-41a

Free

Membrane-

bound

Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)


Quantity of SER and ER Dependent Upon Cell Type

RER assoc. w/ protein synthesis

SER assoc. lipid biosynthesis, detoxification, steroid synthesis, Ca2+ storage


Import of Proteins into ER

►Occurs co-translationally

►Signal recognition sequence recognized by SRP

►SRP recognized by SRP receptor

►Protein Translocator


► Hydrophobic signal sequence of different sequence and shape

► SRP lg hydrophobic pocket lined by Met having unbranched flexible side chains

► Binding of SRP causes pause in protein synthesis allowing time for SRP-ribosome complex to bind to SRP receptor


Protein to be imported passes through an aqueous pore in the translocator that is a dynamic structure

►Sec61 protein translocator

►Signal sequence triggers opening of pore

►Translocator pore closes when ribosomes are not present


Some proteins are imported in to ER by a posttranslational mechanism

►Proteins released into cytoplasm

►Binding of chaperone proteins prevents them from folding

►Translocation occurs without ribosomes sealing pore

►Mechanism whereby protein moves through pore is unknown


Signal Sequence is Removed from Soluble Proteins ► Two signaling functions:

1) directs protein to ER membrane

2) serves as “start transfer signal” to open pore

► Signal peptidase removes terminal ER signal sequence upon release of protein into the lumen


Single Pass Transmembrane Proteins

1. N-terminal signal sequence initiates trans-location and additional hydrophobic “stop sequence anchors protein in membrane

2. Signal sequence is internal and remains in lipid bilayer after release from translocator

3. Internal signal sequence in opposite orientation

4. Orientation of start-transfer sequence governed by distribution of nearby chg aa

Multipass Transmembrane Proteins

Multipass Transmembrane Proteins ► Combinations of start- and stop-transfer signals determine topology

► Whether hydrophobic signal sequence is a start- or stop-transfer sequence depends upon its location in polypeptide chain

► All copies of same polypeptide have same orientation

Folding of ER Resident Proteins

► ER resident proteins contain an ER retention signal of 4 specific aa at C-terminus

► PDI (protein disulfide isomerase) oxidizes free SH grps on cysteines to from disulfide bonds S-S allowing proteins to refold

► BiP chaperone proteins, pulls proteins posttranslationally into ER through translocator and assists w/ protein folding

Glycolsylation of ER Proteins

► Most soluble and transmembrane proteins made in ER are glycolsylated by addition of an oligosaccharide to Asn

► Precursor oligosaccharide linked to dolichol lipid in ER mem, in high energy state

► Transfer by oligosaccharyl transferase occurs almost as soon as polypeptide enters lumen


Retrotranslocation ► Improperly folded ER proteins are exported and degraded in cytosol

► Misfolded proteins in ER activate an “Unfolded Protein Response” to increase transcription of ER chaperones and degradative enzymes


The Unfolded Protein Response

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