Endomembrane System Notes
The Endomembrane System
Eukaryotic cells rely on intercellular membranes for compartmentalization of function within organelles.
The movement of lipids and proteins between organelles, known as trafficking, requires tight regulation.
Components of the Endomembrane System
The endomembrane system includes:
Endoplasmic reticulum (ER)
Golgi complex
Endosomes
Lysosomes
The ER and Golgi complex are sites for protein synthesis, processing, and sorting.
Endosomes carry and sort material brought into the cell.
Lysosomes digest ingested material and unneeded cellular components.
Endoplasmic Reticulum (ER)
Two basic kinds of ER differing in structure and function:
Rough ER: Characterized by ribosomes on the cytosolic side of the membrane.
Transitional elements (TEs) are a subdomain of rough ER that form transition vesicles to shuttle lipids and proteins to the Golgi.
Smooth ER: Lacks ribosomes and is involved in processing and storing non-proteins.
Rough and Smooth ER Distinction
Rough ER membranes form large flattened sheets.
Smooth ER membranes form tubular structures.
Transitional elements of the rough ER resemble smooth ER.
The lumenal spaces of rough and smooth ER are continuous.
Variation in Amounts
Both types of ER are present in most eukaryotic cells, but the relative amounts vary.
Cells involved in the synthesis of secretory proteins have prominent rough ER networks.
Cells producing steroid hormones tend to have extensive networks of smooth ER.
Rough ER Functions
Involved in:
Initial steps of adding carbohydrates to glycoproteins
Folding of polypeptides
Assembly of multimeric proteins
Proteins that are incorrectly folded, modified, or assembled are recognized and exported for degradation.
Smooth ER Functions
Involved in drug detoxification, carbohydrate metabolism, calcium storage, and steroid biosynthesis.
Primarily involved in processing or storing non-protein molecules within cells.
Drug Detoxification
Often involves hydroxylation.
Adding hydroxyl groups to hydrophobic drugs increases their solubility, making them easier to excrete from the body.
Hydroxylation is catalyzed by a member of the cytochrome P-450 family of proteins, also called monooxygenases.
Carbohydrate Metabolism
Smooth ER plays a role in the catabolism of liver glycogen.
Calcium Storage
The sarcoplasmic reticulum of muscle cells is an example of smooth ER specializing in calcium storage.
The ER lumen contains high concentrations of calcium-binding proteins.
Calcium ions are pumped into the ER by ATP-dependent calcium ATPases and are released when needed for muscle contraction.
Steroid Biosynthesis
Cholesterol, cortisol, and steroid hormones share a four-ring structure but differ in the number and arrangement of carbon side chains and hydroxyl groups.
Hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) is the committed step in cholesterol biosynthesis.
It is found in smooth ER of liver cells and is targeted by cholesterol-lowering drugs called statins.
Membrane Lipid Biosynthesis
Smooth ER is the primary source of membrane lipids in eukaryotic cells, with a few exceptions.
Mitochondria synthesize phosphatidylethanolamine.
Peroxisomes synthesize cholesterol.
Chloroplasts contain enzymes for chloroplast-specific lipids.
The Golgi Apparatus
Structure
Consists of a series of flattened membrane-bounded cisternae.
A series of cisternae, usually three to eight, is called a Golgi stack.
Some cells have one large stack, while others, especially secretory cells, have hundreds or thousands of stacks.
It has two networks:
Cis-Golgi network (CGN)
Trans-Golgi network (TGN)
Between the TGN and CGN are medial cisternae, where much of the processing of proteins occurs.
Each compartment is biochemically and functionally distinct, containing specific receptor proteins unique to each portion of the network.
Proteins and lipids leave the Golgi in transport vesicles that continuously bud from the tips of the TGN.
Models for Movement Through the Golgi
Two models explain the movement of lipids and proteins from the Golgi:
The stationary cisternae model
Each cisterna in the Golgi stack is a stable structure.
Transport of materials from one cisterna to another is mediated by shuttle vesicles that bud off from one cisterna and fuse with the next in a cis-to-trans sequence.
The cisternal maturation model
Roles of the ER and Golgi Apparatus in Protein Processing
Protein processing in the ER and Golgi apparatus includes:
Protein folding
Quality control
Glycosylation, the addition of carbohydrate side chains to proteins to form glycoproteins. Recall N- or O-linked glycosylation.
Protein Folding and Quality Control in the ER
Polypeptides fold into their final shapes and assemble with other polypeptides to form multisubunit proteins in the ER lumen.
Molecular chaperones facilitate these folding and assembly events.
Disulfide Bonds
Protein disulfide isomerase is an enzyme that leads to the formation (and breakage) of disulfide bonds between cysteines.
Disulfide bonds begin forming before synthesis of the polypeptide is complete, allowing for the formation and breakage of disulfide bonds until the most stable arrangement can be found.
Protein Quality Control Mechanisms
The unfolded protein response (UPR) uses sensor molecules in the ER membrane to detect misfolded proteins.
The sensors activate signaling pathways that shut down the production of protein except for those required for protein folding and degradation.
ER-associated degradation (ERAD) recognizes misfolded or unassembled proteins and exports them to the cytosol to be degraded by proteasomes.
Glycosylation
Initial glycosylation occurs in the ER.
The initial steps of N-glycosylation take place on the cytosolic surface of the ER membrane.
Later steps take place in the ER lumen.
All carbohydrate side chains initially have a common core oligosaccharide consisting of two units of N-acetylglucosamine, nine mannose units, and three glucose units.
Cotranslational Glycosylation
The oligosaccharide is added to the recipient protein as the polypeptide is being synthesized.
Two ER proteins, calnexin (CNX) and calreticulin (CRT), bind to monoglycosylated glycoproteins and promote proper folding.
Roles of the ER and Golgi Apparatus in Protein Trafficking
Proteins synthesized in the rough ER must be directed to a variety of locations.
Once a protein reaches its destination, it must be prevented from leaving.
Each protein contains a specific “tag” targeting it to a transport vesicle that will take it to the correct location.
A tag may be an amino acid sequence, a hydrophobic domain, oligosaccharide side chain, or some other feature.
Tags can also exclude material from certain vesicles.
Nuclear Gene to Synthesized Polypeptide Review
Tagged polypeptides encoded by nuclear genes begin with transcription of DNA into RNAs.
These RNAs are processed in the nucleus (becoming mRNAs) and then transported to the cytoplasm.
mRNAs associate with free ribosomes in the cytoplasm and translation begins.
Shortly after translation begins, two pathways for sorting the synthesizing polypeptide diverge.
Two Pathways for Sorting Tagged Polypeptides
Pathway for polypeptides destined for the cytosol, mitochondria, chloroplast, peroxisome, and nuclear interior:
Free ribosomes synthesize these polypeptides.
After translation, the polypeptide is released and remains in the cytosol, or an organelle takes it up.
Posttranslational import is the uptake of completed polypeptides with special targeting signals into the organelle.
Pathway for polypeptides destined for the endomembrane system or export from the cell:
The ribosome synthesizing these polypeptides attach to the ER membrane.
The polypeptide is transferred across (or for integral membrane proteins, inserted into) the ER membrane as synthesis occurs. This is called cotranslational import.
These proteins can be directed to the ER, Golgi apparatus, endosomes, and lysosomes. There is also a mechanism to maintain the protein in these locations. Other proteins are incorporated into the plasma membrane or released outside the cell.
Cotranslational Import
Cotranslational import allows some polypeptides to enter the ER as they are being synthesized.
Cotranslational import is the first step in the pathway for delivering proteins to various locations in the endomembrane system.
Proteins trafficked this way are synthesized on free ribosomes which become attached to the ER after translation has begun.
Signal Hypothesis
For polypeptides destined for the ER, the N-terminus contains an ER signal sequence that directs ribosome-mRNA-polypeptide complexes to the surface of the rough ER.
As the polypeptide chain elongates, it progressively crosses the ER membrane and enters the ER lumen.
Proteins containing the ER signal sequences are often called preproteins (prelysozyme or preproinsulin).
They will need to be removed before the finished product will become active.
ER Signaling Sequences
Amino acid sequence is variable but has some unifying factors:
15–30 amino acids long
Three domains: a positively charged N-terminal region, a central hydrophobic region, and a polar region next to the cleavage site.
The ER signaling sequence must be present for the polypeptide to be inserted into or across the membrane.
Mechanism of Cotranslational Import
SRP binds to ER signal sequence and blocks translation.
SRP binds to SRP receptor; ribosome docks on membrane.
GTP binds to SRP and SRP receptor; pore opens as the polypeptide is inserted.
Both GTPs are hydrolyzed and SRP is released.
Signal sequence is cleaved by signal peptidase as polypeptide elongates and translocates into ER lumen.
Completed polypeptide is released into ER lumen, ribosome released, and translocon pore closes.
Protein Routing After ER Entry
Most proteins synthesized on ribosomes attached to the ER are glycoproteins.
Glycosylation and folding occurs in the ER. The glycoprotein is then delivered to the Golgi apparatus for further glycosylation and processing of carbohydrate side chains.
The Golgi apparatus then sorts and distributes the proteins.
Soluble proteins go from the Golgi, to secretory vesicles, to the plasma membrane where the protein is secreted.
But they might possess specific carbohydrate side chains or amino acid sequences that target them from the Golgi to other parts of the endomembrane system.
ER-bound proteins have a specific C-terminal tag.
ER Retention and Retrieval Tags
Protein composition in the ER is maintained by preventing some proteins from escaping the ER and by retrieving others from the Golgi.
Some proteins localized to the ER contain the sequence RXR (Arg-X-Arg; X is any amino acid).
This is a retention tag and is also found in some proteins that are destined for the plasma membrane.
Some proteins returned from the Golgi to the ER contain retrieval tags.
The tags are short C-terminal sequences such as KDEL (Lys-Asp-Glu-Leu) or KXX in mammals and HDEL (His-Asp-Glu-Leu) in yeast.
When a protein with this tag binds a receptor, the receptor-ligand complex is packaged into a transport vesicle for return to the ER.
Golgi-Specific Proteins
All Golgi-specific proteins are integral membrane proteins with one or more membrane-spanning domains.
The length of the hydrophobic domains may determine into which cisternae each protein is incorporated.
The thickness of cellular membranes increases progressively from the ER (5 nm) to the plasma membrane (8 nm).
Correlation of Hydrophobic Domain Length with Golgi Location
The thickness of membranes in the Golgi increases from the CGN to the TGN.
Proteins move from compartment to compartment until the membrane thickness exceeds the length of the transmembrane domains.
This blocks further migration.
Targeting Soluble Lysosomal Proteins
Soluble lysosomal enzymes in the ER and early Golgi compartments undergo N-glycosylation (asparagine) followed by removal of glucose and mannose units.
The mannose residues on the side chains are phosphorylated within the Golgi complex, forming an oligosaccharide containing mannose-6-phosphate.
This tag ensures delivery of lysosomal proteins to the lysosomes.
Multivesicular Endosomes
Degradation and recycling of unneeded or damaged components are carried out by specialized late endosomes called multivesicular endosomes (MVEs), also called multivesicular bodies (MVBs).
Serve as an intermediate between early endosomes and lysosomes, sequestering materials destined for degradation or recycling.
Posttranslational Import as Alternative Mechanism for ER Lumen Import
Some proteins are synthesized in the cytosol and subsequently transported into the ER lumen.
This posttranslational import occurs by a process similar to cotranslational import.
As the protein is synthesized, it associates with chaperones which initially keep the protein unfolded.
Exocytosis and Endocytosis: Transporting Material Across the Plasma Membrane
Two methods (unique to eukaryotes) for transporting materials across the plasma membrane are:
Exocytosis, the process by which secretory vesicles release their contents outside the cell
Endocytosis, the process by which cells internalize external materials
Secretory Pathways
Secretory pathways move proteins from the ER through the Golgi complex to secretory vesicles and secretory granules.
The secretory granules then discharge their contents to the exterior of the cell.
Experiment to Show Secretory Pathways
Proteins were radioactively labeled briefly.
After three minutes, the labeled proteins could be seen primarily in the rough ER.
A few minutes later, the labeled proteins began to appear in the Golgi apparatus.
After 37 minutes, the protein was detected in vesicles budding from the Golgi (named condensing vacuoles by the researchers).
After 117 minutes, the protein began to accumulate in dense zymogen granules, vesicles that discharge their contents out of the cell.
Constitutive Secretion
After budding from the TGN, some vesicles move directly to the cell surface and immediately fuse with the plasma membrane.
This unregulated process is continuous and independent of external signals.
It is called constitutive secretion; one example is mucus secretion by the intestinal lining.
Constitutive secretion was once thought to be a default pathway for proteins synthesized by rough ER.
It was thought that proteins destined to stay in the endomembrane system must have a tag to avoid constitutive secretion.
Current evidence suggests that some tags may be required for constitutive secretion.
Regulated Secretion
Secretory vesicles involved in regulated secretion accumulate in the cell and fuse with the plasma membrane only in response to specific signals.
An important example is neurotransmitter release.
Regulated secretory vesicles form by budding from the TGN as immature secretory vesicles.
The mature secretory vesicles move close to the site of secretion and remain there until receiving a signal. The signal triggers vesicles to release their contents by fusion with the plasma membrane.
is a common trigger.
Polarized Secretion
In many cases, exocytosis of specific proteins is limited to a specific surface of the cell.
For example, intestinal cells secrete digestive enzymes only on the side of the cell that faces into the intestine.
This is called polarized secretion.
Exocytosis
In exocytosis, proteins in a vesicle are released to the exterior of the cell as the vesicle fuses with the plasma membrane.
Animal cells secrete hormones, neurotransmitters, mucus, milk proteins, and digestive enzymes this way.
Plant and fungal cells secrete enzyme and structural proteins for the cell wall.
Process of Exocytosis
Vesicles containing products for secretion move to the cell surface.
The membrane of the vesicle fuses with the plasma membrane.
Fusion with the plasma membrane discharges the contents of the vesicle.
The membrane of the vesicle becomes part of the cell membrane.
The inner (lumenal) surface of the vesicle becomes the outer (extracellular) surface.
Mechanism of Exocytosis
The mechanism of the movement of exocytic vesicles to the cell surface is not clear.
Evidence points to the involvement of microtubules in vesicle movement.
Vesicle movement stops when cells are treated with colchicine, a microtubule assembly inhibitor.
Role of Calcium in Triggering Exocytosis
Fusion of regulated secretory vesicles with the plasma membrane is generally triggered by an extracellular signal.
In most cases, a hormone or neurotransmitter binds receptors on the cell surface and triggers a second messenger inside the cell.
A transient elevation in appears to be an essential step in the signaling cascade.
Endocytosis
Most eukaryotic cells carry out one or more forms of endocytosis for uptake of extracellular material.
Process
A small segment of the plasma membrane folds inward.
It pinches off to form an endocytic vesicle containing ingested substances or particles.
Endocytic Vesicles
Most endocytic vesicles develop into early endosomes, which fuse with vesicles from the TGN.
They acquire digestive enzymes and form new lysosomes.
In phagocytosis, solid particles are ingested.
In pinocytosis, liquids are taken up.
Functions of Endocytosis
Maintaining normal cell homeostasis.
Bringing in nutrients.
Recycling cell receptors.
Protecting from bacterial invasion.
Communication with other cells.
Viruses and Endocytosis
Some viruses:
Co-opt endocytic machinery to enter the cell.
Replicate their genome within the endocytic vesicle.
Encode fusion proteins that allow vesicles to open up and release the virus in the cytoplasm.
Phagocytosis
The ingestion of large particles up to and including whole cells or microorganisms.
For many unicellular organisms, it is a means of acquiring food.
For more complex organisms, it is usually restricted to specialized cells called phagocytes.
In humans, two types of white blood cells use phagocytosis as a means of defense:
Neutrophils and macrophages engulf and digest foreign materials or invasive microorganisms found in the bloodstream or injured tissues.
Macrophages are also scavengers, ingesting cellular debris and damaged cells.
Phagocytosis in the Amoeba
Triggered by contact with food or other particles, forming pseudopods (folds of membrane).
These surround the object and engulf it, forming an intracellular phagocytic vacuole (phagosome).
The phagosome fuses with a late endosome or matures directly into a lysosome.
In the lysosome, the engulfed material is digested.
Receptor-Mediated Endocytosis (Clathrin-Dependent Endocytosis)
Cells acquire some substances by receptor-mediated endocytosis.
Cells use receptors on the outer cell surface to internalize many macromolecules.
Mammalian cells can ingest hormones, growth factors, serum proteins, enzymes, cholesterol, antibodies, iron, viruses, bacterial toxins.
Process
Specific molecules (ligands) bind to their receptors on the outer surface of the cell.
Receptor-ligand complexes diffuse laterally and encounter specialized regions called coated pits, sites for collection and internalization of these complexes.
In a typical mammalian cell, coated pits occupy about 20% of the total surface area.
Accumulation of receptor-ligand complexes in the pits triggers the accumulation of additional proteins on the cytosolic surface of the membrane.
These proteins—adaptor protein, clathrin, and dynamin—induce curvature and invagination of the pit.
The pit pinches off, forming a coated vesicle.
The clathrin coat is released, leaving an uncoated vesicle.
The uncoated vesicle fuses with an early endosome.
Transport vesicles often:
Carry material to the late endosome and then a lysosome for digestion.
Recycle receptors to the plasma membrane.
Transport material to the plasma membrane on the opposite side of the cell (transcytosis).
Recycling Plasma Membrane Receptors
Receptors from the plasma membrane are recycled as a result of the acidification of the early endosome.
The pH gradually declines as the endosome matures, facilitated by an ATP-dependent proton pump.
The lower pH dissociates ligand and receptors, allowing receptors to be returned to the membrane.
Alternative Fates for Receptor-Ligand Complexes
Some complexes are carried to a lysosome for degradation.
Some complexes are carried to the TGN, where they enter a variety of pathways in the endomembrane system.
Complexes can also travel by transport vesicles to a different region of the plasma membrane, where they are secreted (transcytosis).
Lysosomes and Cellular Digestion
The lysosome is an organelle of the endomembrane system that contains digestive enzymes.
It can degrade all the major classes of biological macromolecules:
Lipids, carbohydrates, nucleic acids, and proteins
Lysosome Properties
Lysosomes contain acid phosphatase and several other hydrolytic enzymes.
They vary in size and shape but are usually about 0.5 µm in diameter, bounded by a single membrane.
The lumenal side of the membrane is coated with glycoproteins to protect the membrane from degradation.
Acidity
Lysosomes maintain an acidic environment (pH 4.0–5.0) inside.
ATP-dependent proton pumps in the membrane are responsible for this.
There are numerous enzymes inside lysosomes; all are acid hydrolases.
Lysosome Development
Lysosomal enzymes are synthesized by ribosomes on rough ER and translocated inside.
Lysosomal enzymes are delivered from the TGN to endosomes in transport vesicles.
Over time, endosomes mature into late endosomes, with all the enzymes, but not engaged in digestion.
Endosome Development
The pH of the early endosome lumen drops from about 6.0 to 5.5.
The endosome loses its ability to fuse with endocytic vesicles.
The late endosome is packaged with material to be digested and newly synthesized digestive enzymes.
The last step in the development of a lysosome is the activation of the acid hydrolases.
This occurs as the internal environment becomes more acidic (pH 4.0–5.0).
This occurs through the pumping of protons or through fusion with an existing lysosome.
Types of Lysosomes and Digestive Properties
Lysosomes containing substances that originated outside the cell (phagocytosis and receptor-mediated endocytosis) are called heterophagic lysosomes.
Those with materials that originated inside the cell (worn out, broke-down cell parts) are called autophagic lysosomes.
Fate of Indigestible Material
Eventually, indigestible material is all that remains in a lysosome.
The lysosome becomes a residual body when digestion ceases.
Some cells release the contents by exocytosis. In others, accumulation of debris may contribute to cellular aging.
Autophagy: A Biological Recycling System
Cellular structures that are damaged or unneeded must be broken down via autophagy.
Extracellular Digestion
Most lysosomal digestion occurs inside the cell.
In some cases, lysosomes discharge their contents outside the cell, resulting in extracellular digestion.
For example, the head of a sperm releases digestive enzymes to degrade barriers protecting an egg.
Plant Vacuoles
Plant cells have acidic vacuoles that perform the function of lysosomes, but with additional roles.
Vacuole development is similar to that of lysosomes, with coated vesicles conveying materials for the vacuole to a provacuole, similar to an endosome.
Additional Functions of Vacuoles
Vacuoles mature to form a structure that can fill up to 90% of the cell’s total volume.
They maintain turgor pressure, the osmotic pressure preventing plant cells from collapsing.
They regulate cytosolic pH, using ATP-dependent proton pumps.
Vacuoles serve as a storage compartment for such substances as:
Seed storage proteins
The anion Malate (in CAM plants)
Anthocyanins
Toxic components to deter predators
Soluble and insoluble waste
Peroxisomes
Peroxisomes are bounded by single membranes thought to be supplied by the ER.
The defining characteristic of peroxisomes is the presence of catalase for degrading .
Peroxisome Structure
Animal peroxisomes contain a crystalline core, consisting of crystalline urate oxidase.
Plant peroxisomes may contain crystalline catalase.
Peroxisomes without the crystalline cores may be difficult to identify microscopically.
Peroxisomal Functions
Essential roles include:
Hydrogen peroxide metabolism
Detoxification of harmful compounds
Oxidation of fatty acids
Metabolism of nitrogen-containing compounds
Catabolism of unusual substances
Hydrogen Peroxide Metabolism
The dollar signs (RH2+O2
ightharpoonup R + H2O2H2O2H2O2 + H2O2
ightharpoonup 2H2O + O2H2O2RH2 + H2O2 ightharpoonup R + 2H2O$$ (Catalase as a peroxidase)
Detoxification of Harmful Compounds
Peroxidase can use methanol, ethanol, formic acid, formaldehyde, nitrates, and phenols (all toxic) as electron donors. This oxidative detoxification protects the cell.
Peroxisomes detoxify reactive oxygen species.
Peroxisomal enzymes such as superoxide dismutase and others detoxify reactive oxygen species.
Oxidation of Fatty Acids
Peroxisomes contain enzymes for β oxidation of fatty acids.
In animals, the primary product of β oxidation, acetyl-CoA, is exported to the cytosol and enters biosynthetic pathways or the citric acid cycle.
In plants and yeast, fatty acids are completely oxidized in peroxisomes. A certain type of lipid, plasmalogen, which is crucial to the myelinization of neurons, is created in peroxisomes. A defect in peroxisomes leads to neurological problems
Metabolism of Nitrogen-Containing Compounds
Most animals (not primates) require urate oxidase to oxidize urate, formed during catabolism of nucleic acids and some proteins.
Aminotransferases
Aminotransferases catalyze the transfer of amino groups from amino acids to α-keto acids in the degradation and synthesis of amino acids.
Catabolism of Unusual Substances
D-amino acids are rare substances for which the cell has no degradative pathways, except in the peroxisome.
In some cells, peroxisomes also contain enzymes that break down xenobiotics, compounds foreign to living organisms.
This includes short-chain hydrocarbons such as alkanes.
Glyoxysomes
Glyoxysomes occur transiently in seedlings.
They contain enzymes needed to convert stored triacylglycerols to sucrose, β oxidation of fatty acids, and the glyoxylate cycle.
They are found only in tissues where fat is stored, and when no longer needed, they are converted to peroxisomes.
Plant-Specific Peroxisomes
In plants and algae, peroxisomes are involved in several aspects of cell energy metabolism.
Cells of photosynthetic tissues contain leaf peroxisomes in close contact with mitochondria and chloroplasts.
They are involved in the glycolate pathway, also called the photorespiratory pathway.