CBNS101 Lecture 05 Transmembrane transport into the ER and mitochondria
Page 1: Introduction to Cell Biology
Fundamentals of Cell Biology:
Focus on transmembrane transport into the endoplasmic reticulum (ER) and mitochondria.
Credit to Riccardo Cassiani-Ingoni / Science Photo Library.
Page 2: Overview of Transmembrane Transport
Transmembrane transport of proteins into the endoplasmic reticulum (ER):
Pathways include:
Nucleus
Cytosol
Plastids
Peroxisomes
Mitochondria
Endoplasmic Reticulum
Golgi apparatus
Secretory vesicles
Late endosomes
Lysosomes
Transport mechanisms:
Gated transport
Vesicular transport
Engulfment by the plasma membrane and cell exterior.
Page 3: The Endoplasmic Reticulum (ER)
Significance of ER:
Present in all eukaryotic cells.
Accounts for over 50% of the total membrane space in an average animal cell.
Functions:
Synthesis and modification of lipids for cellular membranes.
Synthesis and modification of proteins; initial step to the secretory pathway.
Gateway for proteins destined for Golgi apparatus, lysosomes, or secretion outside of the cell.
Storage site for Ca2+ ions to maintain low cytosolic Ca2+ levels.
Page 4: Structure of the ER
The ER forms a continuous network of branching sheets and tubules.
Close proximity of ER membranes to all regions of the cytosol.
Distribution influenced by microtubules.
Fluorescence micrograph showcases a protein retained in the ER.
Page 5: Types of ER
Rough ER:
Coated with membrane-bound ribosomes, involved in protein synthesis and translocation into the ER.
Smooth ER:
Lacks ribosomes, serves as sites for transport vesicle budding and enzymatic catalysis.
Page 6: Interactions with Other Organelles
Smooth ER regions can communicate with other organelles for metabolite (e.g., lipid) transfer.
Contacts can occur between ER, mitochondrion, and plasma membrane.
Page 7: Signal Sequences and Transport
Discovery of signal sequences in proteins imported into the ER.
Hypothesis of signal sequence's association with ribosomes for target proteins.
Work by Blobel, Sabatini, and Dobberstein (1971) linked targeting information to the amino-terminus of proteins.
Dr. Guenter Blobel awarded the Nobel Prize in Medicine (1999) for his contributions to protein targeting.
Page 8: Key Observations by Blobel
mRNA for a secreted protein translated in vitro in cytoplasmic extracts with/without ER microsomes.
Observed variations in protein size and translocation based on presence or absence of microsomes.
Microsomes formed from broken ER are critical for proper protein processing.
Page 9: Co-Translational Transfer Model
Experiments supported the co-translational transfer model for proteins exported outside the cell.
Recognition of signal sequence on ribosome.
Formation of a seal and translocation of peptide across the ER membrane.
Cleavage of the signal sequence during this process.
Page 10: Role of Signal Recognition Particle (SRP)
SRP directs ER signal sequences to their specific receptor in the ER membrane.
Composed of six proteins and a small RNA molecule.
SRP homologs are conserved entities across different organisms, indicating early evolution.
Page 11: Variability of ER Signal Sequences
Signal sequences exhibit variability in amino acid sequences but contain over eight nonpolar amino acids at their center.
The plasticity of the binding pocket helps accommodate diverse hydrophobic signal sequences.
Page 12: Structuring of SRP
SRP is rod-like with a signal-binding end that binds to the signal during translation.
The other end wraps around the ribosome, impacting translation speed.
Binding exposes a receptor site on SRP for the SRP receptor in the ER membrane.
Page 13: Translocation Dynamics
SRP binding brings ribosome to the protein translocator to assist in protein transfer into the ER.
Continuation of translation allows proteins to enter the ER lumen.
Signal peptidase cleaves the signal sequence following successful translocation.
Page 14: Populations of Ribosomes
Distinction between:
Free ribosomes in the cytosol
Membrane-bound ribosomes.
Polyribosomes involve multiple ribosomes translating an identical mRNA.
Page 15: Role of Sec61 Complex
The Sec61 complex forms the protein translocator channel.
It has a signal-sequence-gated channel that opens in the presence of an appropriate signal sequence.
The entry and exit dynamics of proteins, especially hydrophobic ones, is crucial to their integration.
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Page 17: Post-Translational Translocation
Occurs in yeast ER and bacterial plasma membranes.
Proteins remain unfolded in the cytosol due to chaperones prior to translocation.
Retrotranslocation is common for proteins destined for degradation.
Page 18: Transmembrane Proteins in the ER
All transmembrane proteins are initially inserted into the ER membrane.
Involves segments integrated into the membrane and others retained in the cytosol.
Requires machinery like SRP, SRP receptor, and Sec61 translocator for effective transport.
Page 19: Insertion Mechanism for Single-Pass Transmembrane Proteins
Recognized by SRP as internal signal sequences.
Orientation (N-terminus inside or outside the ER) is determined by the characteristics of the transmembrane segment.
Properties include stability of the N-terminal part and net charge.
Page 20: N-Terminal Domains and Translocation
For proteins intended to remain in the ER lumen, N-terminal sequences initiate translocation and folding.
Transmembrane segments exit through the Sec61 complex, halting translocation for the hydrophobic region.
Page 21: Multiple-Pass Transmembrane Proteins
Each transmembrane segment must be inserted in an alternating orientation.
Translocation through Sec61 is diligently managed with no cleavage for the subsequent segments post-initial insertion.
Page 22: GPI-Anchored Proteins
Made with typical signal sequences, most enter the ER lumen before a hydrophobic sequence near the C-terminus accepts a GPI anchor.
Commonly results in N-terminal domains facing outward on the plasma membrane.
Page 23: ER Function Summary
The ER plays a critical role in lipid production, protein processing, and calcium storage.
Signal sequences are essential for protein movement, aiming for the rough ER and through the Sec61 translocator.
Orientation for transmembrane proteins is defined by their first segment, with GPI anchoring being common.
Page 24: Protein Folding and Modification in ER
Proteins arrive in an unfolded state and require assistance to fold.
BiP protein chaperone:
Prevents aggregation by binding to hydrophobic regions.
PDI (Protein Disulfide Isomerase):
Forms disulfide bonds to stabilize proteins and corrects misfolded ones.
Page 25: Glycosylation in ER
Improperly folded proteins undergo glycosylation—majority N-linked.
Oligosaccharides are added to nascent proteins, becoming glycoproteins, with extensive modifications.
N-linked modifications are more common than O-linked.
Page 26: Degradation of Misfolded Proteins
Misfolded proteins are identified and targeted for retrotranslocation.
Misfolded markers include oligosaccharide trimming and binding by lectins.
Transport to the cytosol is ATP-driven; substrate proteins are ubiquitinated for degradation.
Page 27: Unfolded Protein Response (UPR)
UPR activation occurs due to misfolded protein accumulation.
Three pathways (IRE1, PERK, ATF6) stimulate transcription of genes to manage protein folding.
PERK: - Reduces general translation.
ATF6: Moves to the nucleus to activate response genes.
Page 28: Additional UPR Pathways
IRE1:
Responds to misfolded protein presence; activates itself and splices XBP1 mRNA for a transcription factor.
UPR also plays role in managing normal ER load during increased protein synthesis, like insulin production.
Page 29: Lipid Bilayers Synthesized in ER
New lipids form on the cytosolic side of the ER membrane.
Scramblases promote equal distribution, while flippases help maintain asymmetry.
Page 30: ER Membrane Contact Sites
Mitochondria and chloroplasts obtain lipids from the ER via non-vesicular transport.
Lipid transfer occurs at membrane junctions utilizing binding proteins.
Page 31: Folding and Glycosylation Summary
Proteins fold in the ER with help from chaperones.
Improperly folded proteins are sent back to the cytosol for degradation through ubiquitin tagging.
UPR pathways address the accumulation of misfolded proteins while regulating lipid synthesis.
Page 32: Transport Mechanisms in Mitochondria and Chloroplasts
Overview of transmembrane transport anatomy for mitochondria and chloroplasts.
Page 33: Unique Transport Needs of Mitochondria and Chloroplasts
Both organelles are double-membrane bound with distinct roles and requirements for nuclear-encoded proteins.
Page 34: Challenges in Mitochondrial Protein Transport
Various proteins target multiple mitochondrial compartments, necessitating diverse transport strategies and signal sequences.
Page 35: Signal Sequences in Mitochondrial Transport
TOM complex manages protein recognition and routing.
Proteins may have signal sequences targeting various mitochondrial sites, like inner membrane or matrix.
Page 36: Mitochondrial Transport Mechanisms
Import of proteins occurs post-translationally, utilizing TOM and TIM translocators based on specific sequences.
Page 37: Unfolded State in Mitochondrial Import
Cytosolic precursor proteins remain unfolded thanks to chaperones until recognition by TOM.
Translocation dynamics are similar to those in ER but require different energy sources.
Page 38: Energy Dynamics of Mitochondrial Import
Import involves ATP hydrolysis and membrane potential effects to drive protein translocation through inner membrane.
Page 39: Complex Pathways for Membrane Proteins
Transmembrane proteins utilize various mechanisms for appropriate insertion into mitochondrial membranes.
Page 40: Proteins Synthesized Within Mitochondria
Fully translocated proteins can subsequently expose additional sequences that target them to the inner membrane via OXA.
Page 41: Insertion of β-Barrel Proteins
Hydrophobic segments exit TOM and insert into OM via SAM complex for folding and stability.
Page 42: Chloroplast Transport Mechanisms
Chloroplast transport parallels mitochondrial processes but requires distinct signal sequences for targeting specific membranes like thylakoids.
Page 43: Summary of Mitochondrial and Chloroplast Transport
Most proteins for these organelles are cytoplasm-imported.
Utilize multiple translocator complexes to manage distinct transport pathways, emphasizing energy and structural mechanisms.
Structure and Function of the Endoplasmic Reticulum (ER)
Present in all eukaryotic cells, accounting for over 50% of total membrane space in an average animal cell.
Functions include the synthesis and modification of lipids and proteins, acting as a gateway for proteins destined for various organelles or secretion, and serving as a storage site for Ca2+ ions.
Co-Translational Transfer of Proteins into the ER
Proteins recognized by ribosomes enter the ER simultaneously during translation, with the signal sequence directing their entry.
This involves forming a seal and translocating the peptide across the ER membrane, cleaving the signal sequence during this process.
Roles of SRP, SRP Receptor, and Sec61
Signal Recognition Particle (SRP): Directs ER signal sequences to the specific receptor in the ER membrane, composed of proteins and RNA.
SRP Receptor: Binds to SRP which has brought the ribosome to the ER, facilitating the docking and translocation process.
Sec61 Complex: Functions as the protein translocator channel with a gated channel that opens upon the presence of an appropriate signal sequence, allowing proteins to enter the ER lumen.
Insertion of Proteins into ER Membrane
Single-Pass Transmembrane Proteins: Recognized by SRP as internal signal sequences; orientation determined by transmembrane segment characteristics.
Multiple-Pass Transmembrane Proteins: Each segment is inserted in an alternating orientation through the Sec61 complex without cleavage after the initial insertion.
Non-Vesicular Lipid Transfer to Mitochondria and Chloroplasts
Mitochondria and chloroplasts obtain lipids from the ER through non-vesicular transport at membrane contact sites, utilizing binding proteins for lipid transfer.
Post-Translational Transport of Proteins into Mitochondria and Chloroplasts
Involves proteins remaining unfolded in the cytosol, with chaperones preventing aggregation until translocation occurs through distinct translocators after translation is complete.
Signal Sequences and Translocators in Targeting Proteins
Signal sequences guide proteins to various mitochondrial sites, with TOM (Translocase of the Outer Membrane) and TIM (Translocase of the Inner Membrane) ensuring proper localization based on specific sequences.
Function of Chaperone Proteins
In the ER, chaperones like BiP prevent aggregation by binding to hydrophobic regions of polypeptides. In mitochondria, chaperones also assist in maintaining proteins in an unfolded state until they reach their target compartments.
Practice Test Questions (Multiple Choice)
Structure and Function of the ERa. What percentage of total membrane space in an average animal cell is occupied by the endoplasmic reticulum (ER)?
A) 25%
B) 50%
C) 75%
D) Over 50%
b. Which of the following is NOT a function of the ER?
A) Synthesis of proteins
B) Storage of Ca2+ ions
C) Photosynthesis
D) Lipid modification
Co-Translational Transfera. What role does the signal sequence play in the co-translational transfer of proteins into the ER?
A) Initiates protein folding
B) Directs the entry of proteins into the ER
C) Stabilizes proteins in the cytosol
D) Alters the protein's amino acid sequence
Roles of SRP, SRP Receptor, and Sec61a. What is the primary function of the Signal Recognition Particle (SRP)?
A) To fold proteins
B) To direct ER signal sequences to their receptor
C) To cleave signal sequences
D) To transport proteins across the membrane
b. What does the Sec61 complex do?
A) Synthesizes lipids
B) Serves as a protein translocator channel
C) Modifies glycoproteins
D) Acts as a ribosomal subunit
Insertion of Proteins into ER Membranea. How do single-pass transmembrane proteins differ from multiple-pass transmembrane proteins?
A) They are always larger in size
B) Single-pass proteins have one transmembrane segment, while multiple-pass proteins have two or more
C) Single-pass proteins do not require SRP
D) Multiple-pass proteins are only found in mitochondria
b. What determines the orientation of transmembrane proteins?
A) The length of the signal sequence
B) The characteristics of the transmembrane segment
C) The type of ribosome translating the protein
D) The presence of chaperones
Lipid Transfer to Mitochondria and Chloroplastsa. How do mitochondria and chloroplasts receive lipids from the ER?
A) Through vesicular transport
B) Via non-vesicular transport at membrane contact sites
C) By diffusion through the cytosol
D) Through the Golgi apparatus
Post-Translational Transporta. What is a characteristic of proteins during their post-translational transport into mitochondria and chloroplasts?
A) They remain folded
B) They become modified during transport
C) They remain unfolded and are assisted by chaperones
D) They are tagged for degradation
Signal Sequences and Translocatorsa. What is the function of TOM in mitochondria?
A) It folds proteins
B) It translocates proteins across the outer membrane
C) It modifies lipids
D) It cleaves signal sequences
Chaperone Proteinsa. How do chaperone proteins aid protein folding in the ER?
A) By adding carbohydrates to proteins
B) By preventing aggregation and assisting with folding
C) By cleaving misfolded proteins
D) By translocating proteins into the membrane
b. What is the role of chaperones in mitochondrial protein transport?
A) They facilitate ATP synthesis
B) They maintain proteins in an unfolded state until targeting is achieved
C) They modify proteins for function
D) They aid in the synthesis of new proteins
Note
Each question has multiple choice options to test your understanding of the concepts related to the structure and function of the endoplasmic reticulum, co-translational transfer, protein targeting, and chaperone proteins.