Class 14: Intracellular Compartments and Protein Transport

Membrane-Enclosed Organelles and Protein Sorting

  • Eukaryotic cells utilize membrane-enclosed organelles to compartmentalize various cellular processes.
  • Key organelles include:
    • Mitochondria
    • Golgi apparatus
    • Endoplasmic reticulum (ER) with membrane-bound polyribosomes
    • Endosomes
    • Lysosomes
    • Cytosol
    • Peroxisomes
    • Free ribosomes
    • Nucleus
    • Plasma membrane

Strategies for Organizing Cellular Processes

  • Cells employ two main strategies to organize biochemical reactions:
    • Aggregation into Multicomponent Complexes: Enzymes required for a particular sequence of reactions are assembled into a single complex. Examples include DNA and RNA synthesis and ribosome assembly.
    • Compartmentalization via Membrane-Enclosed Organelles: Eukaryotic cells confine different metabolic processes and proteins into membrane-bound compartments.

Eukaryotic Organelles

  • Nucleus
    • Enclosed by a double membrane called the nuclear envelope, which is perforated by nuclear pores.
    • The outer nuclear membrane is continuous with the ER membrane.
  • Endoplasmic Reticulum (ER)
    • A system of interconnected membranous sacs and tubes extending throughout much of the cell.
    • Major site of new membrane synthesis.
  • Smooth Endoplasmic Reticulum (Smooth ER)
    • Lacks ribosomes.
    • Involved in:
      • Synthesis of most lipids.
      • Steroid hormone synthesis in some endocrine cells of the adrenal gland.
      • Detoxification of alcohol and other organic molecules in liver cells.
      • Sequestration of Ca2+Ca^{2+} from the cytosol, regulating muscle contraction and responses to extracellular signals.
    • Proliferation of smooth ER in response to toxins (e.g., barbiturates) due to increased activity of detoxification enzymes like cytochrome P450.
    • Proliferation leads to drug tolerance as cells become better equipped to modify drugs.
  • Rough Endoplasmic Reticulum (RER)
    • Ribosomes attached to its cytosolic surface actively synthesize proteins.
    • These proteins are either inserted into the ER membrane or delivered to the ER lumen.
  • Golgi Apparatus
    • Usually located near the nucleus.
    • Receives proteins and lipids from the ER.
    • Modifies and sorts these proteins and lipids.
    • Dispatches them to other destinations within the cell.
  • Lysosomes
    • Small sacs containing digestive enzymes.
    • Degrade worn-out organelles, macromolecules, and particles taken into the cell by endocytosis.
  • Endosomes
    • A series of compartments through which endocytosed materials pass en route to lysosomes.
    • Sort ingested molecules.
    • Recycle some molecules back to the plasma membrane.
  • Peroxisomes
    • Small organelles containing oxidative enzymes.
    • Break down lipids and destroy toxic molecules using O2O_2.
    • Produce H<em>2O</em>2H<em>2O</em>2 and use it in oxidation reactions.
    • Catalase enzyme converts excess H<em>2O</em>2H<em>2O</em>2 to H<em>2OH<em>2O and O</em>2O</em>2: 2H<em>2O</em>2→2H<em>2O+O</em>22 H<em>2O</em>2 \rightarrow 2 H<em>2O + O</em>2
  • Mitochondria
    • Surrounded by a double membrane.
    • Sites of oxidative phosphorylation and ATP production.
    • Contain internal membranes specialized for ATP production.
    • Contain their own mtDNA and ribosomes.

Intracellular Membranes and Compartmentalization

  • Internal membranes create enclosed compartments within eukaryotic cells, separating different metabolic processes.
  • Example: Liver cells contain aggregates of glycogen and enzymes that control its synthesis and breakdown.

Organelle Volume and Number

  • Membrane-enclosed organelles occupy nearly half the volume of a eukaryotic cell.
  • Example: In a liver cell (hepatocyte):
    • Cytosol: 54% of cell volume, ~1 per cell
    • Mitochondria: 22% of cell volume, ~1700 per cell
    • ER: 12% of cell volume, ~1 per cell
    • Nucleus: 6% of cell volume, ~1 per cell
    • Golgi apparatus: 3% of cell volume, ~1 per cell
    • Peroxisomes: 1% of cell volume, ~400 per cell
    • Lysosomes: 1% of cell volume, ~300 per cell
    • Endosomes: 1% of cell volume, ~200 per cell

Organelle Membrane Surface Area

  • Organelle membranes are extensive.
  • Example: In a typical mammalian cell, the area of the ER membrane is 20-30 times greater than that of the plasma membrane.
  • The plasma membrane is a minor membrane in most eukaryotic cells.

Evolution of Membrane-Enclosed Organelles

  • Archaea and Bacteria:
    • Can function with just a plasma membrane due to their small size.
    • Have a sufficient surface area-to-volume ratio.
  • Eukaryotic Cells:
    • Volumes are 1000-10,000 times higher than prokaryotes.
    • The plasma membrane does not provide enough functional membrane for such a high cellular volume.
    • Internal membranes increase the surface area-to-volume ratio.
    • Except for mitochondria and chloroplasts, organelles likely evolved via invagination of the plasma membrane.

Endomembrane System and Evolutionary Origin

  • In bacteria and archaea, DNA is attached to the plasma membrane.
  • In ancient anaerobic archaeon, the plasma membrane with attached DNA may have invaginated.
  • Over generations, this formed a two-layered envelope surrounding the DNA.
  • This envelope pinched off, creating a nuclear compartment with nuclear pores for communication with the cytosol.
  • Invaginated membrane may have also formed the ER, explaining why the space between the inner and outer nuclear membranes is continuous with the ER lumen.

Endomembrane System

The interiors of organelles within the system are treated as extracellular. The nucleus has two membranes.
The components are:

  • ER
  • Golgi apparatus
  • Peroxisomes
  • Endosomes
  • Lysosomes

Interiors of these organelles communicate with one another and the cell exterior via vesicles that bud off from one organelle and fuse with another.

Protein Sorting - Overview

  1. Proteins are transported into organelles via 3 mechanisms.
  2. Signal sequences direct proteins to the correct compartment.
  3. Mechanism 1: Proteins enter the nucleus through nuclear pores.
  4. Mechanism 2: Proteins unfold to enter mitochondria.
  5. Proteins enter peroxisomes from both the cytosol and ER.
  6. Proteins enter the ER while being synthesized.
  7. Soluble proteins made on the ER are released into the ER lumen.
  8. Start and stop signals determine the arrangement of transmembrane proteins in the lipid bilayer.
  9. Mechanism 3: Vesicular Transport – Discussed in Class 15

Protein Sorting - Introduction

  • Directing newly synthesized proteins to the correct organelle is crucial for cell function, growth, and division.
  • Before cell division, eukaryotic cells must duplicate their membrane-enclosed organelles.
  • As cells grow, these organelles get bigger and then divide.
  • During cell division, organelles are distributed between two daughter cells.

Protein Delivery

  • Proteins are delivered directly from the cytosol to:
    • Mitochondria
    • Chloroplasts
    • The interior of the nucleus
    • ER
    • Peroxisomes (bulk of digestive enzymes from cytosol)
  • Proteins and lipids are delivered indirectly via the ER to:
    • Golgi apparatus
    • Lysosomes
    • Endosomes
    • Inner nuclear membrane
    • Peroxisomes

Protein Entry

  • Proteins enter the ER directly from the cytosol.
    • Some are retained in the ER.
    • Most are transported to the Golgi by vesicles and then onward to the plasma membrane or other organelles.
  • The ER is also a major site of protein and lipid synthesis.

Address Labels

  • Address labels are contained in amino acid sequences.
  • Once at the correct address, proteins enter the membrane or interior lumen of the organelle.

Protein Transport Mechanisms

  • Virtually all proteins in the cell begin synthesis on ribosomes in the cytosol, except for a few synthesized within mitochondria and chloroplasts.
  • The fate of a protein synthesized in the cytosol depends on its amino acid sequence, which may contain a sorting signal directing it to the appropriate organelle.
  • If no sorting signal is present, the protein remains in the cytosol.
  • Different sorting signals direct proteins to the nucleus, mitochondria, peroxisomes, and ER.
  • Hydrophilic, soluble proteins must cross hydrophobic bilayers to enter organelles.

Mechanisms

  • Mechanism 1: Cytosol ⇄ Nucleus
  • Mechanism 2: Cytosol → ER, Cytosol → Mitochondria
  • Mechanism 3: ER → other compartments of the endomembrane system, Compartments of the endomembrane system → other compartments of the endomembrane system

Protein Translocation and Nuclear Pores

  • Proteins are transported across organelle membranes by protein translocators; they usually must unfold to pass through.
  • Nuclear Pores – selective gates that actively transport specific macromolecules and also allow free transport of smaller molecules
  • Proteins are transported through nuclear pores, which penetrate both inner and outer nuclear membranes.
  • Proteins are transported by transport vesicles, which pinch off from the membrane of one compartment and fuse with the membrane of another.
  • Transport vesicles deliver soluble proteins, membrane proteins, and lipids.

Signal Sequences

  • A typical signal sequence is a continuous stretch of 15-60 amino acids.
  • The signal sequence is often cleaved off the finished protein after sorting.

Nuclear Import

  • The inner nuclear membrane contains proteins that act as binding sites for chromosomes and provide anchorage for the nuclear lamina.

Nuclear Pores

  • Nuclear pores perforate the double membrane of the nuclear envelope.
  • The outer nuclear membrane closely resembles the ER membrane and is continuous with it.
  • Ribosomes are normally bound to the cytosolic surface of the ER membrane and the outer nuclear membrane.
  • Protein fibrils protrude from both sides of the pore complex, forming a basket-like structure on the nuclear side.

Nuclear Pore Complex

  • The nuclear pore complex consists of approximately 30 different proteins, each present in multiple copies.
  • Many proteins lining the nuclear pore form a soft, tangled meshwork that fills the center of the channel, preventing the passage of large molecules but allowing small water-soluble molecules to pass freely.
  • Protein fibrils protrude from both sides of the pores.
  • RNA molecules and ribosomal subunits are exported to the cytosol.
  • Newly synthesized proteins destined for the nucleus are imported through nuclear pores.

Gaining Entry to the Nucleus

  • To enter the pore, proteins must display a nuclear localization signal.
  • Nuclear import receptor proteins in the cytosol recognize the nuclear localization signal.
  • Receptors direct newly synthesized proteins to the pore by interacting with tentacle-like fibrils extending from the pore rim into the cytosol.
  • Nuclear import receptors disrupt interactions between nuclear pore proteins, clearing a path through the pore's interior meshwork.
  • Receptor proteins return through the pore for reuse.
  • Nuclear export receptors function similarly, driving protein and RNA traffic from the nucleus to the cytosol, recognizing nuclear export signals.
  • Proteins are transported into the nucleus in a fully folded conformation.
  • Energy supplied by GTP hydrolysis drives nuclear transport.

Mitochondrial Import

  • Chaperone proteins inside mitochondria help pull proteins across membranes and fold them once inside.
  • Subsequent sorting directs proteins to the correct part of the mitochondria.
  • Insertion of transmembrane proteins into the inner membrane is guided by signal sequences that start and stop the transfer process across the membrane.
  • Most mitochondrial membrane phospholipids are imported from the ER.

Protein Import into Mitochondria

  • Most mitochondrial proteins are encoded by nuclear DNA and synthesized in the cytosol.
  • These proteins usually have a signal sequence at the N-terminus that allows them to enter mitochondria.
  • The signal sequence is removed after translocation is complete.
  • Proteins are simultaneously translocated across both inner and outer membranes at specialized sites where the two membranes are closely apposed.
  • Each protein unfolds as it is transported.

Mitochondrial Translocation

  1. The mitochondrial signal sequence on a mitochondrial precursor protein is recognized by a receptor in the mitochondrial outer membrane (OM).
  2. The receptor is associated with a protein translocator, which transports the signal sequence across the OM to the intermembrane space.
  3. The complex of receptor, precursor protein, and translocator diffuses laterally in the OM until the signal sequence is recognized by a second translocator in the inner membrane (IM).
  4. Together, the two translocators transport the protein across both the OM and IM, unfolding the protein in the process.
  • The signal sequence is cleaved off by a signal peptidase in the mitochondrial matrix.
  • Mitochondrial precursor proteins are unfolded during import.

Peroxisomal Import

  • Peroxisomes are packed with enzymes that:
    • Digest toxins.
    • Synthesize certain phospholipids, including those in the myelin sheath surrounding nerve cell axons.
  • Peroxisomes mainly get proteins from the cytosol by selective transport.
    • A short sequence of three amino acids is the import signal for many peroxisomal proteins.
    • The peroxisomal membrane has a translocator that aids in protein transport, but proteins do not need to unfold to enter the peroxisome.
    • A few proteins embedded in the peroxisomal membrane arrive via vesicles that bud off from the ER.

Peroxisomes - Clinical Implication

  • Zellweger syndrome is caused by mutations that block peroxisomal protein import, leading to severe abnormalities in the brain, liver, and kidneys. Most affected individuals do not survive past the first 6 months, demonstrating the crucial role of peroxisomes and peroxisomal protein transport in life.

Endoplasmic Reticulum (ER)

  • The most extensive membrane system in a eukaryotic cell.
  • Up to 50-90% of the total membrane in a mammalian cell surrounds the ER lumen.
  • It forms a continuous network of flattened sacs (cisternae), tubules, and associated vesicles throughout the cytoplasm of eukaryotic cells.
  • ER cisternae are membrane-bounded sacs.
  • The ER lumen is the space enclosed within the ER cisternae.

ER - Site of Protein Synthesis

  • The site of synthesis of proteins destined for:
    • Plasma membrane
    • Organelles of the endomembrane system
    • Secretion by the cell
    • Central role in lipid synthesis

Protein translocation across the ER membrane

  1. Water-soluble proteins are completely translocated across the ER membrane and released into the ER lumen; they are either secreted or transported to the lumen of another organelle.
  2. Prospective transmembrane proteins are partly translocated across the ER membrane and become embedded in it; they stay in the membrane of the plasma membrane or one of the organelles of the endomembrane system.
    *

  • The entry point for proteins destined for the ER and for proteins destined for other organelles or the plasma membrane via transport vesicles.

  • Different from transport across membranes of other organelles because transport starts before polypeptide chain has been completely synthesized.

  • The ribosome synthesizing protein is attached to the ER membrane (RER).

  • The outer nuclear membrane, which is continuous with the ER, is also studded with ribosomes.

Ribosomes in the Cytosol

  • Two populations of ribosomes in the cytosol:
    1. Membrane-bound ribosomes are attached to the cytosolic side of the ER membrane and the outer nuclear membrane, making proteins being translocated into the ER.
    2. Free ribosomes make all the other proteins encoded by nuclear DNA.

Protein translocation requirements

  • Proteins with an ER sequence are being translocated as they are synthesized, so no additional energy is needed for transport across the ER membrane.
  • Elongation of each polypeptide provides the thrust needed to push the growing chain through the ER membrane.
  • The ER signal sequence, consisting of 8 or more hydrophobic amino acids, directs the protein to the ER.
  • As the mRNA molecule is translated, many ribosomes bind to it, forming a polyribosome.
  • If the proteins being synthesized have an ER signal sequence, the polyribosome attaches to the ER membrane.

Protein Guidance to the ER Membrane

  • Three protein components help guide ER signal sequences to the ER membrane:
    1. Signal recognition particle (SRP)
    2. SRP receptor
    3. Protein translocator
  • The signal recognition particle (SRP) in the cytosol binds to the ribosome and the ER signal sequence.
  • The SRP receptor, which is embedded in the ER membrane, recognizes the SRP.
  • Once bound to the SRP receptor, the SRP is released, and the receptor passes the ribosome to the protein translocator in the ER membrane.
  • The polypeptide is threaded across the ER membrane through a channel in the protein translocator.
  • The signal sequence opens the protein translocator.
  • The signal sequence stays bound to the translocator while the rest of the polypeptide chain is threaded through the membrane as a large loop.
  • The signal sequence is removed by a transmembrane signal peptidase.
  • Once protein synthesis is complete, the translocated polypeptide is released as a soluble protein into the ER lumen.

Transmembrane Proteins

  • Some proteins made by ribosomes attached to the ER remain embedded in the ER membrane as transmembrane proteins.
  • Some parts of the protein are completely translocated, while other parts stay in the lipid bilayer.

Start-Transfer and Stop-Transfer Sequences

  1. A signal sequence at the beginning initiates translocation.
  2. A stop-transfer sequence further along the polypeptide chain stops the transfer process.

Single-Pass Transmembrane Proteins

  • A single-pass transmembrane protein has an N-terminal signal sequence.
  • The N-terminal signal sequence is cleaved off by signal peptidase.
  • The stop-transfer sequence remains in the bilayer.
  • Both sequences are hydrophobic.

Double-Pass Transmembrane Proteins

  • A double-pass transmembrane protein has an internal signal sequence.
  • The signal sequences are not cleaved off but stay and anchor the protein in the membrane.
  • If a protein spans the membrane more than two times, additional pairs of start- and stop-transfer sequences repeat the process for each pair. The start-transfer sequence initiates the transfer, and the stop-transfer sequence halts it, resulting in parts of the polypeptide located on both sides of the membrane. This process is repeated as often as necessary.