Endoplasmic Reticulum and Membrane Proteins

Lecture 2: The Endoplasmic Reticulum (ER)

Introduction

• The endoplasmic reticulum (ER) is a crucial organelle in eukaryotic cells, playing a central role in lipid and protein biosynthesis.

• The ER is organized into a network of branching tubules and flattened sheets, forming a reticulum-like structure.

• A portion of the ER, known as the transitional ER, is responsible for transporting molecules to the Golgi apparatus.

Toxins and the Lipid Bilayer

Cholera Toxin

• The lipid bilayer is the site of action for certain toxins.

• Ganglioside GM1 acts as a cell-surface receptor for cholera toxin in intestinal epithelial cells.

• Entry of cholera toxin leads to increased intracellular cyclic AMP (cAMP) levels, resulting in massive secretion of Na^+, K^+, HCO_3^-, and water into the intestinal lumen, causing severe diarrhea.

• Water and electrolyte infusion can save most patients.

• Mechanism:

  • Cholera toxin (CTx) holotoxin binds to the plasma membrane via its pentameric B subunit.

  • It is internalized and delivered to endosomes.

  • The toxin enters the retrograde trafficking pathway, reaching the ER.

  • The A subunit is released from the B subunit and unfolded by protein disulfide isomerase (PDI).

  • The A subunit translocates across the ER membrane into the cytoplasm.

  • The A subunit then refolds, activating adenylate cyclase (AC), leading to increased cAMP levels.

  • This triggers chloride secretion and subsequent watery diarrhea.

• Cholera toxin is also a tool to study membrane trafficking.

Pore-Forming Toxins

• Many pathogens produce pore-forming toxins that damage the cell membrane, resulting in leakage and lysis of target cells.

• These toxins are hydrophilic proteins rich in β-sheets (β-PFTs).

• β-PFTs bind to a cell surface receptor, oligomerize into a β-barrel structure, insert into the lipid bilayer, and create a channel.

• The pore sizes vary (e.g., 15-30 Å to 250-300 Å).

ER Structure and Function

General Features

• All eukaryotic cells have an ER.

• The relative importance of the ER's various functions varies greatly between individual cell types.

• In cells that produce proteins, the ER constitutes >1/2 of the total membrane in the cell.

• The ER and Golgi lumens are topologically equivalent to the exterior of the cell.

• Polypeptide chains in the ER lumen will be exposed on the cell surface after transport to the plasma membrane.

• Polypeptide chains in the cytoplasm will remain in the cytoplasm.

• The tubules interconnect and are continuous with the nuclear membrane, forming a continuous sheet enclosing a single internal space called the ER lumen, forming the secretory pathway.

Sorting Signals

• Sorting signals are specific amino acid sequences recognized by sorting receptors.

• The receptors function catalytically, returning to their origin for reuse after each targeting round.

• Most sorting receptors recognize classes of proteins rather than individual protein species.

• The Golgi apparatus receives lipids and proteins from the ER and dispatches them to various destinations using transport vesicles, often modifying them en route (e.g., glycosylation).

Rough ER (RER) vs. Smooth ER (SER)

• Rough ER (RER):

  • Combination of sacs and cisternae with ribosomes, giving it a rough appearance.

  • Synthesizes proteins destined for the cell surface (or ER, lysosomes, Golgi, or secretion).

• Smooth ER (SER):

  • More tubular appearance with no ribosomes, giving it a smooth appearance.

  • Synthesizes lipids, including cholesterol and phospholipids, for new cell membranes.

• Each cell contains both SER and RER in different amounts depending on the cell specialization.

• More protein production implies more RER, while more detox, lipid synthesis, or Ca^{2+} storage implies more SER.

Microsomes

• When cells are disrupted, the ER fragments into small vesicles called microsomes.

• Rough microsomes derived from the RER are lined with ribosomes on their outer surface and can be separated from smooth microsomes via equilibrium density-gradient centrifugation.

Communication with the Nucleus

• The rough ER is in close proximity with the nucleus.

• It can rapidly send feedback to the nucleus when problems in protein synthesis and folding occur (UPR), influencing the overall rate of protein translation.

Smooth ER Functions

• Detoxification:

  • Contains enzymes to detoxify lipid-soluble small molecules (drugs) and harmful compounds produced by metabolism.

  • Cytochrome P450 enzymes catalyze reactions to transform water-insoluble drugs or metabolites into water-soluble molecules for excretion.

  • They metabolize thousands of endogenous and exogenous chemicals, preventing accumulation of metabolites to toxic levels.

  • For example, bilirubin is transported to the smooth ER for conjugation with glucuronic acid.

• Lipid Synthesis:

  • Synthesizes the majority of cellular lipids and cholesterol.

  • The ER has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species.

• Calcium Storage:

  • Sequestering and storing Ca^{2+} from the cytosol.

  • A lot of Ca^{2+}-binding proteins (e.g. calsequestrin, calmodulin, etc.)

  • Storage in the ER provides the release of Ca^{2+} into the cytosol and its subsequent reuptake occurs in many rapid responses to extracellular signals.

  • Muscle cells have an abundant smooth ER called the sarcoplasmic reticulum.

  • The release and reuptake of Ca^{2+} by the sarcoplasmic reticulum trigger myofibril contraction and relaxation.

Rough ER Functions

• Site of production of all transmembrane proteins for most organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane.

• Site of folding, assembly of oligomers, and post-translational modification (PTM) of all of these proteins.

• Almost all proteins secreted by the cell and those destined for the lumen of the ER, Golgi apparatus, or lysosomes are made in the ER lumen and then modified in the Golgi apparatus.

Membrane Proteins

General Characteristics

• Membrane proteins perform most of the membrane’s specific tasks, giving each cell membrane its characteristic functional properties.

• In myelin membranes (electrical insulation), <25% of the membrane mass is protein, while in membranes involved in ATP production (e.g., mitochondria, chloroplasts), ~75% of the mass is protein.

• Membrane proteins are amphiphilic, with hydrophobic regions passing through the membrane and hydrophilic regions exposed to water on either side.

• Only transmembrane proteins can function on both sides of the bilayer or transport molecules across it.

• Cell-surface receptors are usually transmembrane proteins that bind ligands in the extracellular space and transduce intracellular signals.

• How a membrane protein is associated with the lipid bilayer is key for the function of the protein

Types of Membrane Proteins

• Single α helix

• Multiple α helices

• Rolled-up β sheet or β barrel

• Anchored to the cytosolic surface by an amphiphilic α helix

• Attached to the bilayer solely by a covalently bound lipid (e.g., palmitic acid, prenyl group, GPI anchor)

Lipid Anchors

• Myristic acid (a saturated 14-carbon fatty acid) is added to the N-terminal amino group of some proteins (Myristoyl anchor).

• Palmitic acid (a saturated 16-carbon fatty acid) is added to a cysteine (Cys) side chain of the protein (Palmitoyl anchor).

• Farnesylation is also a common anchor (Farnesyl anchor).

Membrane Protein Localization

• Cells can confine proteins and lipids to specific domains within a membrane.

• In epithelial cells (e.g., enterocytes or kidney tubules), certain plasma membrane enzymes and transport proteins are confined to the apical or basal/lateral surfaces.

• This asymmetric distribution is often essential for the function of the epithelium (e.g., Na^+ pumping in a specific direction).

• The proteins have a signal peptide, which directs the protein into the ER where they fold

Post-Translational Modifications

• N- and O-linked glycosylation (vast majority)

• Disulfide bonds (majority)

• GPI anchor (a small subset)

• Phosphorylation (some of them)

• Transmembrane domain(s) (TMD): 1 (vast majority) to 18 TMDs, with 7 TMD being the second most common (GPCRs)

• Often modular architecture

• They can be targeted by drugs that do NOT penetrate the cell

Cell Surface and Secreted Proteins

• Membrane-bound and secreted proteins are synthesized in the ER and modified in the Golgi

Review

• ER general structural features and difference between RER and SER

• RER and SER overall functions

• Import proteins into the secretory pathway can co- or post-translational

• Overall architecture of the many types of membrane proteins