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Chapter 3

:
Endomembrane
System

PROTEIN BIOCHEMISTRY &
BIOTECHNOLOGY
BTP2223
By: MOHD AZMIR BIN ARIFIN
Contact No.: 019-238127
E-Mail: mazmir@ump.edu.my

OUTLINES

• Basic biology and the structure of endomembrane
system
• Endoplasmic reticulum (ER) structure and functions
• Mechanisms of protein entry into ER lumen, protein
exit from ER, unfolded protein response & protein
degradation

The subcellular complexity of cell

Endomembrane
• Composed of the different membranes that are suspended
in the cytoplasm within a eukaryotic cell.
• Divide the cell into functional and structural
compartments, or organelles.
• In eukaryotes : the nuclear membrane, the endoplasmic
reticulum (ER), the Golgi apparatus, lysosomes,
endosomes and the cell membrane*

carbohydrates. nucleic acids.and exoxytic routes (membrane transport pathway)* – Lysosomes: Degradation processes.• Endomembrane system contains the following components: – Endoplasmic reticulum (ER): • Smooth ER • Rough ER - Golgi complex: Protein modification – Endosomes: Protein sorting between endo. and cellular debris ) – Peroxisomes (Microbodies): beta-oxidation of fatty acids . including proteins. organelle turnover. lipids. (containing hydrolytic enzymes capable of breaking down virtually all kinds of biomolecules.

. Porter.Endoplasmic Reticulum (ER) HISTORY • In the year 1945 -> The lace like membranes of the endoplasmic reticulum were first seen in the cytoplasm of chick embryo cells -> a team of biologists. and Ernest F. Fullman • 1952.ER term used by Porter and Fullman in their published article. Keith R. Albert Claude.

• Highly convoluted space is called the ER lumen or the ER cisternal space. • The ER membrane separates the ER lumen from the cytosol. • It makes up approximately 12% of cell volume.Endoplasmic Reticulum (ER) • Endoplasmic means "within the plasm" and reticulum means "network". . and it mediates the selective transfer of molecules between these two compartments. • A network of tubular and flat vesicular structures in the cytoplasm is the endoplasmic reticulum • The tubules and vesicles interconnect with one another • It’s an internal delivery system of cell.

• ER membrane are constructed of lipid bilayer that contain large amounts of proteins. • Electron micrographs show that the space inside the endoplasmic reticulum is connected with the space between the two membrane surfaces of the nuclear membrane (perinuclear space) • Also it is connected with Golgi apparatus and cell membrane .STRUCTURE • Endoplasmic matrix: The space inside the tubules and vesicles is filled with a watery medium that is different from the fluid in the cytosol outside the ER. similar to the cell membrane.

.

for instance-can be as much as 30 to 40 times the cell membrane area.STRUCTURE • All eukaryotic cells have an ER except the red blood cells and spermatozoa • The total surface area of this structure in some cellsthe liver cells. • The functions of the endoplasmic reticulum vary greatly depending on its cell type. • The ER can even modify to change over time in response to cell needs. cell function. and cell needs. .

TYPES • 2 types: Rough ( granular) endoplasmic reticulum (RER) Smooth ( agranular) endoplasmic reticulum (SER) • The quantity of RER and SER in a cell can slowly interchange from one type to the other. depending on changing metabolic needs. .

• acinar cell of pancreas* • White blood cells that produce infection fighting immune system proteins called antibodies have highly developed RER • Ribosome: large numbers of minute granular particles attached to the outer surfaces of many parts of the rough endoplasmic reticulum. .eg.RER • Rough ER is well developed in cells that are active in protein synthesis.

containing many different proteins and at least three ribosomal RNAs. the 60S and 40S subunits. • Each is made up of a large and a small subunit called. • They are the sites of protein synthesis . on the basis of their rates of sedimentation in the ultracentrifuge* • The ribosomes are complex structures.RER • The ribosomes in eukaryotes measure approximately 22 x 32 nm.

• Free ribosomes are also found in the cytoplasm. • The binding site of the Ribosome on RER is the translocon formed by the heterotrimeric Sec61 complex*.RER • These proteins typically have a hydrophobic signal peptide at one end. .

• The ribosomes bound to the RER at any one time are not a stable part of this organelles structure as ribosomes are constantly being bound and released from the membrane.RER • The free ribosomes synthesize cytoplasmic proteins such as hemoglobin and the proteins found in peroxisomes and mitochondria. and most proteins that are stored in the Golgi apparatus. and endosomes. most secreted proteins. • The ribosomes that become attached to the endoplasmic reticulum synthesize all transmembrane proteins. lysosomes. .

• They are not permanently attached. adhering only when there is protein manufacturing work to be done .• Rough ER appears as lumpy sheets of folded membranes. the lumps are the ribosomes.

• The SER consists of tubules that are located near the cell periphery. .SER • Part of the endoplasmic reticulum has no attached ribosomes. • These tubes sometimes branch forming a network that is reticular in appearance. • This part is also called the agranular endoplasmic reticulum.

phospholipids and steroids). • SER is the site of lipid synthesis (including oils. regulation of calcium concentration and detoxification of drugs and poisons. .SER • The network of SER allows increased surface area for the action or storage of key enzymes and the products of these enzymes. metabolizing of carbohydrates.

• Smooth ER found in smooth and striated muscle. • In brain cells it synthesizes male and female hormones. .SER • It is found in abundance with Leydig cell and cells of adrenal cortex*.

. while the SR stores and pumps calcium ions*. • The only structural difference between this organelle and the smooth ER is the medley of proteins they have. • This fundamental difference is indicative of their functions -> The ER synthesizes molecules. smooth ER is modified to form sarcoplasmic reticulum. ("flesh“)* • In skeletal and cardiac muscle. from the Greek sarx. both bound to their membranes and drifting within the confines of their lumens.SARCOPLASMIC RETICULUM • The sarcoplasmic reticulum (SR).

The RER gives rise to the smooth ER by loss of ribosomes. .The RER produce enzyme precursors for the formation of lysosomes by Golgi Complex. • Smooth ER formation.Linking of sugars to glycoprotein starts in the RER and is completed in Golgi complex. quality control and export of completed proteins • Formation of Glycoprotein. • Synthesis of precursors.FUNCTION RER : • Predominant function is in the import of nascent polypeptides into lumen* • Involves largely in folding.

lysosomes and vacuoles. and skin oil glands have a great deal of smooth endoplasmic reticulum. converts harmful materials(drugs. carbohydrate metabolism. i. • Formation of organelles - The SER produces Golgi apparatus . • Detoxification-The SER brings about detoxification in the liver .  and detoxification  and is especially abundant in mammalian liver and gonad cells. Cells which secrete these products. • It also synthesizes  phospholipids.e. .FUNCTION SER : • The smooth endoplasmic reticulum lacks ribosomes and functions in lipid metabolism.. such as those in the testes. poisons) into harmless ones for excretion by the cell. ovaries.

which converts glucose-6phosphate to glucose. . • In muscle cells. it regulates calcium ion concentration (SR) • The smooth endoplasmic reticulum also contains the enzyme glucose-6-phosphatase.FUNCTION SER : • It also carries out the attachment of receptors on cell membrane proteins and steroid metabolism. a step in gluconeogenesis (carbohydrate  metabolism).

FUNCTION SR : • Specializes in the storage of calcium • Calcium ions are pumped into the ER by ATP-dependent calcium ATPases and are released in response to extracellular signals to aid in muscle contraction • Binding of neurotransmitter molecules to receptors on the surface of the muscle cell triggers a signal cascade that leads to the release of calcium from the sarcoplasmic reticulum and causes the contraction of muscle fibers. .

Internal transport pathway in cells Gated transport* • Via protein channels or pores • Eg: nuclear import and export Transmembrane transport • Also known as translocation • Via protein translocation channel • Eg: Protein entry into the ER Vesicular transport • Via membrane-bound intermediates (vesicles) • Eg: ER to Golgi .

Overview of sorting of nuclear-encoded proteins in eukaryotic cells mRNAs are translated on cytosolic ribosomes Ribosomes synthesizin g ER proteins Proteins without ER signal are sent to cytosol Those proteins with organellesp uptake sqn Nascent proteins are will be sent to directed to several RER by the signal sqn destinations Translation completed here Proteins moved to golgi complex via vesicles from whence they are transported to several destinations Partially folded protein Fully folded Fully folded .

. in collaboration with David Sabatini and Bernhard Dobberstein of Rockefeller University proposed a theory They suggested the following: • many proteins have a signal sequence at their N-terminus that functions like a postal code for the target organelle*. Günter Blobel. • Post-translational translocation (translocation after the process of translation is complete). • If the synthesized proteins "belong" in a different organelle.Protein translocation In the early 1970s. they can be transported there in either of two ways depending on the protein: • Co-translational translocation (translocation during the process of translation). • The translation of mRNA into protein by a ribosome takes place within the cytosol.

As the signal sequence emerges from the ribosome. The nascent polypeptide passes through a protein-lined pore in the ER membrane and into the ER lumen. Step 3. Step 2. it is bound by a signal recognition particle (SRP) that arrests further synthesis.Synthesis of Secretory. Step 4. The SRP is released from the membrane. or Plant Vacuolar Proteins on Membrane-Bound Ribosomes Step 1. Lysosomal. . This mediates the binding of the complex to the RER membrane by the SRP receptor.

The signal sequence allows the recognition particle to bind to the ribosome causing the ribosome to bind to the RER pass the new protein through the ER membrane Signal sequence is cleaved by signal peptidases release the new PP into the ER lumen .

Co-translational entry into the ER • Many proteins are made on ribosomes attached to cytosolic surface of RER membranes • Nascent polypeptides attached to ribosomes are directed to the ER through interaction of a ‘signal sequence’ on the new protein with a cytosolic molecule termed ‘signal recognition particle’ (SRP) • A typical signal sequence: +H3N-Met-Met-Ser-Phe- • Val-Ser-Leu.Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-ThrGlu-Ala-Glu-Gln-LeuThr-Lys-Cys-Glu-Val-Phe-Gln (hydrophobic. SRP is composed of 6 distinct polypeptides and a positively small 7Scharged) RNA molecule. negatively charged. Its binding briefly halts translation until the complex is brought to a translocon channel on the RER membrane* .

• Leaving the protein free in the lumen of the ER.Co-translational entry into the ER (cont’d) SOLUBLE PROTEINS • The peptide moves through the translocation channel into the lumen of the ER. • It is later cleaved off by a signal peptidase. • The signal peptide sequence remains attached to the membrane. .

This orientation of the protein persists all of the way to its final destination. That is. The hydrophobic transmembrane domain holds the protein in the membrane because of the very strong Import of a membrane protein. The blue sheath-like component shown in the figure is the transport complex that moves the protein through the membrane. . As membrane proteins are being translated. This example is a single pass membrane protein that contains a single membrane crossing domain. the cytosolic side of membrane remains on the cytosolic side throughout all processes. This serves as a 'stop transfer' signal and leaves the protein inserted in the ER membrane. This figure illustrates the case of a protein being incorporated in the membrane of the endoplasmic reticulum.Co-translational entry into the ER (cont’d) MEMBRANE PROTEINS • • • Key point is that the orientation of a protein in the membrane is established when it is first inserted into the membrane. they are translocated or transferred into the ER until a hydrophobic membrane crossing domain is encountered.

however. are imported into the ER after their synthesis has been completed. the translocator needs accessory proteins that feed the polypeptide chain into the pore and drive translocation . . • To function in post-translational translocation.• Post-translation translocation Some proteins.

two membrane-bound proteins. This pathway is poorly understood in eukaryotes.Cotranslational targeting of secretory proteins to the ER Posttranslational translocation of proteins into the ER In prokaryotes this requires certain cofactors such as SecA and SecB. but is facilitated by Sec62 and Sec63. .

Sec63 recruits the luminal chaperone Kar2 to the luminal face of the membrane where it is suggested to promote the unidirectional movement of the protein being imported by acting as a molecular ratchet*.Post-translational translocation of secretory proteins at the yeast ER. . Hsp70 chaperones maintain the translocation competence of post-translational precursors prior to them encountering the ER membrane. The polypeptide binds the Sec61/62/63/71/72 complex and inserts into the ER translocation channel.

BACTERIA vs ARCHEA vs EUCARYOTES .

oligosaccharide is held in the ER lumen by the lipid called dolichol* • The oligosaccharide is transferred to the emerging protein by the enzyme oligosaccharyl transferase (OST) . proteins are glycosylated on arginine residues (N-Linked) • The precursor.Protein folding and glycosylation in the ER • About half of all eukaryotic proteins are glycosylated • Almost all proteins entering the secretory pathway are glycosylated • Immediately on entering the ER lumen.

Protein Glycosylation in the RER .

such as calnexin (transmembrane) and calreticulin (soluble) • Disulphide bonds are formed with the help of protein disulphide isomerase (PDI) • The glucosyl transferase (GT) enzyme is able to recognise the unfolded proteins .Quality control in the ER • Glycosylation is used as a marker for the state of protein folding* • The first phase involves the trimming of the two terminal residues • The proteins (now with a single terminal glucose) undergo folding while bound to molecular chaperones.

. trapping the protein in the ER. • Removal of the terminal glucose by a glucosidase releases the protein from calnexin.• The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides. renewing the protein's affinity for calnexin and retaining it in the ER. • The cycle repeats until the protein has folded completely. the enzyme transfers a new glucose from UDP-glucose to the Nlinked oligosaccharide. • A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded.

The role of glycosylation in the ER protein folding .

Exiting the ER – misfolded proteins • Despite all the help from chaperones. where it is degraded* . many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve their properly folded state. where they are degraded • Once the misfolded protein has reached the cytosol. its oligosaccharides are removed. • Such proteins are exported from the ER back into the cytosol. • Deglycosylation is catalyzed by an N-glycanase. which removes the oligosaccharide chains by cleaving the amide bond between the carbonyl group and the amino group of the original asparagine to which the oligosaccharide was attached. • The deglycosylated polypeptide is rapidly ubiquitylated by ER-bound ubiquitin-conjugating enzymes and is then fed into proteasomes.

The export & degradation of misfolded ER protein .

the N-glycanase enzyme removes the carbohydrate side chain (oligosaccharide chains ) • The small ubiquitin (8.• Proteins that are consistently unable to be folded are eventually ‘dislocated’ from the ER in a process termed ER-associated degradation (ERAD) • There is strong evidence that the unfolded protein passess back through the translocon into the cytosol • In the cytosol.5 kDa) is added to the dislocated proteins to mark them for degradation by the proteosome .