Protein Localization Flashcards
Membrane-enclosed organelles often have characteristic positions in the cytosol
Protein pathway from nucleus
Nucleus
ER
Transport vesicle
Golgi Apparatus (Cis face)
Golgi Apparatus (Trans face)
Secretory vesicle
Protein expelled
Function of proteins are determined by their localizations in cells
- Cytosolic Protein Synthesis
Proteins are synthesized by ribosomes in the cytosol.
Some proteins remain in the cytosol as they lack specific localization signals.
- Endoplasmic Reticulum (ER) Targeting
Proteins with an ER signal sequence are directed to the rough ER.
The signal sequence allows entry into the ER lumen, where they undergo folding and modifications.
- Golgi Complex Processing
Proteins from the ER are transported to the Golgi apparatus.
The Golgi further processes and sorts proteins for their final destinations.
- Secretory Pathway
Some proteins are sent to the plasma membrane for secretion or become integral membrane proteins.
Others are targeted to lysosomes for degradation functions.
- Peroxisomal Targeting
Proteins with a peroxisomal targeting sequence are directed to peroxisomes.
Peroxisomes play roles in lipid metabolism and detoxification.
- Mitochondrial and Chloroplast Targeting
Proteins meant for mitochondria or chloroplasts contain specific targeting sequences.
These sequences allow them to be imported into their respective organelles, where they function in energy metabolism (e.g., ATP production in mitochondria, photosynthesis in chloroplasts).
Conclusion
The localization of proteins is determined by specific signal sequences within their structure.
Proper localization is essential for cellular function, as proteins need to be in the correct organelles or membranes to perform their roles efficiently.
4 ways proteins are transported:
1.Protein translocation
*Protein translocators transport proteins
*E.g. cytosol-ER lumen, mitochondria, ER
2.Gated transport
*Involves selective gates
*E.g. nuclear transport
3. Vesicular transport
*Involves transport vesicles
4. Engulfment/autophagy
*involves of formation of compartments
PGVE
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Nucleus <-> Cytosol
Gated transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Cytosol -> Plastids
Protein translocation
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Cytosol -> Mitochondria
Protein translocation
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Cytosol -> ER
Protein translocation
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Cytosol -> Peroxisomes
Protein translocation
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
ER -> Peroxisomes
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
ER <-> Golgi Body
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Golgi Body <-> Late Endosome
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Golgi Body <-> Early Endosome
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Golgi Body -> Plasma membrane and Cell exterior
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Golgi Body <-> Secretory vesicles
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Late Endosome -> Lysosome
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Secretory Vesicles -> Plasma membrane and Cell exterior
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Cytosol -> Lysosome
Engulfment
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Early Endosome -> Late Endosome
Vesicular transport
A simplified “roadmap” of protein traffic
Identify which type of protein transport:
Plasma membrane and Cell exterior <-> Early Endosome
Vesicular transport
2 ways in which sorting signals can be built into a protein:
Signal sequence
Signal patch
- a continuous stretch of amino acid sequence, typically 15-60 residues long that resides in a single discrete stretch of amino acid sequence
- used to direct proteins from the cytosol into the ER, mitochondria, chloroplasts, and peroxisomes, and nucleus to the cytosol and from the Golgi apparatus to the ER
- Recognized by sorting receptors
Signal sequence
Where is the signal sequence located in an amino acid:
Amino group or Carboxyl group?
Amino group
- 3D arrangement of amino acids formed by the juxtaposition of amino acids from regions that are physically separated before the protein folds.
- Cytosol-nucleus
Signal patch
Each signal sequence specifies a particular destination in the cell
Import to nucleus
-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-
Export from nucleus
-Met-Glu-Glu-Leu-Ser-Gln-Ala-Leu-Ala-Ser-Ser-Phe-
Import into mitochondria
N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-
Transport of molecules between Nucleus and Cytosol
__________ occurs continuously between the cytosol and the nucleus
Bidirectional traffic
- encloses the DNA and defines the nuclear compartment.
Nuclear envelope
Nuclear envelope has two membranes
inner nuclear membrane
Outer nuclear membrane
contains specific proteins that act as binding sites for chromatin and for the nuclear lamina
inner nuclear membrane
- space between the inner and outer nuclear membranes which is continuous with ER lumen
perinuclear space
_________
- Made up of protein subunits ________ (cytoskeletal proteins)
- Structural support
- Anchoring site for chromosomes and NPC (Nuclear Pore Complexes)
Nuclear lamina; nuclear lamins
is continuous with the membrane of the ER and studded with ribosomes
Outer nuclear membrane
____________
- Perforated with nuclear core complexes (NPC).
- Each pore complex contains 1 or more open aqueous channels through which small water-soluble molecules can passively diffuse
- Each NPC composed of __________ different proteins, called ___________
Nuclear envelope; more than 30; nucleoporins
a filamentous structure attached to the nucleoplasmic side of the nuclear pore complex (NPC), crucial for regulating transport between the nucleus and cytoplasm, and thought to act as a platform for mRNA quality control and export.
Nuclear basket
key proteins that form the central channel within the nuclear pore complex (NPC), facilitating the transport of molecules between the nucleus and cytoplasm.
Channel nucleoporins
are proteins that form the structural framework of the nuclear pore complex (NPC)
Scaffold nucleoporins
form rings and anchor the NPC to the nuclear envelope, facilitating the transport of molecules between the nucleus and cytoplasm.
Membrane ring proteins
also known as cytoplasmic filaments, are protein structures that extend from the nuclear pore complex (NPC) into the cytoplasm, playing a crucial role in regulating nuclear transport and interacting with the cytoskeleton.
Cytosolic fibrils
RNA Viruses: These viruses must enter the nucleus for replication, often through nuclear pore complexes.
Orthomyxoviridae
Bornaviridae
DNA Viruses: These viruses deliver their genetic material into the nucleus for replication.
Polyomaviridae
Papillomaviridae
Baculoviridae
Reverse Transcribing (RT) Viruses:
ssRNA(RT) Viruses: These integrate into the host genome after reverse transcription.
Lentiviruses
Reverse Transcribing (RT) Viruses:
dsDNA(RT) Viruses: Partially double-stranded DNA viruses that complete their genome in the nucleus.
Hepadnaviridae
ssDNA Viruses: These need the nucleus to convert their single-stranded DNA into a double-stranded form for replication.
Parvoviridae
Circoviridae
Geminiviridae
dsDNA Viruses: These directly deliver double-stranded DNA into the nucleus.
Herpesvirales
Adenoviridae
Different Behavior in Quiescent vs. Dividing Cells:
Quiescent Cells (Non-dividing): Viruses must actively transport their genetic material through nuclear pores.
Dividing Cells: Some viruses (e.g., Retroviridae except Lentivirus) wait until mitosis when the nuclear envelope dissolves, allowing direct access to the host genome.
Nuclear Localization Signals Direct Nuclear Proteins to the Nucleus
- signal sequences or signal patches
- consist of 1 or 2 short sequences that are rich in the positively charged amino acids, ________ and _________
lysine; arginine
Localization of T-antigen containing a mutated nuclear import signal
Pro-Pro-Lys-“Thr”-Lys-Arg-Lys-Val-
__________
- soluble cytosolic proteins that bind both to the nuclear localization signal on the protein to be transported and to nucleoporins.
- To initiate nuclear import, most nuclear localization signals must be recognized by nuclear import receptors (________)
Nuclear import receptors; karyopherins
serve as binding sites for the import receptors
FG (phenylalanine glycine) repeats
a small GTP-binding protein that acts as a molecular switch, regulating the translocation of RNA and proteins through the nuclear pore complex (NPC)
Ran protein
Ran in the Nucleus (Ran-GTP Form)
Inside the nucleus, Ran is bound to GTP (Ran-GTP).
Ran-GTP interacts with nuclear transport receptors to facilitate cargo release or binding.
- Promotes the exchange of GDP for GTP
- Converts Ran-GDP into Ran-GTP
- Triggers GTP hydrolysis and converts Ran-GTP to Ran-GDP
Ran GEF (guanine nucleotide exchange factor); Ran GAP (GTPase-activating protein)
Ran GAP and Ran GEF maintain the Ran cycle, ensuring Ran-GTP is concentrated in the nucleus and Ran-GDP in the cytoplasm.
Nuclear Import
- Protein binds to nuclear import receptor
- Ran-GTP binds to nuclear import receptor
- Protein delivered to nucleus
- GTP is hydrolyzed, Ran-GDP dissociates from receptor
Nuclear Export
- Ran-GTP binds to nuclear export receptor
- Protein with nuclear export signal (cargo) binds to nuclear export receptor with Ran-GTP
- Cargo delivered to cytosol (Ran-GDP+P
Transport Between the Nucleus and Cytosol Can Be Regulated by _______________
Controlling Access to the Transport Machinery
nuclear localization and export signals can be turned on or off, often by _____________
phosphorylation of adjacent amino acids
(High, Low) [Ca2+] in resting T cell
(High, Low) [Ca2+] in activated T cell
Low; High
a protein involved in T-cell activation
Calcineurin (protein phosphate)
MDM2 is a key negative regulator of the p53 tumor suppressor protein, functioning as an E3 ubiquitin ligase that promotes p53 degradation, thereby limiting p53’s growth-suppressive activity.
- Is used to label proteins, antibodies, peptides, hormones, amine-modified oligonucleotides, and other amine-containing molecules with green fluorescent dye (fluorescein)
Fluorescein isothiocyanate (FITC)
Chloroplast structure
Outer membrane
Intermembrane space
Inner membrane
Thylakoid
Granum (Stack of thylakoid)
Stroma
Stroma lamellae
Mitochondria structure
Outer membrane
Inner membrane
Cristae
Matrix
Transport in Mitochondria
- mitochondrial proteins are first fully synthesized as precursor proteins in the cytosol and then translocated into mitochondria by a posttranslational mechanism.
- Most of the mitochondrial precursor proteins have a signal
sequence at their N terminus that is rapidly removed after import.
Protein translocators in the mitochondrial membranes
- TOM complex
- functions across the outer membrane
- TIM complexes,
- TIM23 and TIM22 complexes, function across the inner membrane
- OXA complex
- mediates the insertion of inner membrane proteins that are synthesized within the mitochondria
- helps to insert some proteins that are initially transported into the matrix by the TOM and TIM complexes
Protein translocators in the mitochondrial membranes:
- is required for the import of all nucleus-encoded mitochondrial proteins.
- It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane.
TOM complex
Protein translocators in the mitochondrial membranes:
- transports proteins into the matrix space, while helping to insert transmembrane proteins into the inner membrane.
TIM 23
Protein translocators in the mitochondrial membranes:
- mediates the insertion of a subclass of inner membrane proteins, including the carrier protein that transports ADP, ATP, and phosphate.
TIM22 complex
- Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as ___________.
These precursors contain a ____________ (MTS), which directs them to the mitochondria.
_______________ binds to the precursor protein, preventing premature folding and aggregation.
ATP hydrolysis provides energy to maintain the precursor protein in an import-competent state.
- The mitochondrial outer membrane contains __________ that recognize the matrix-targeting sequence.
The receptor, such as Tom20 or Tom22, facilitates the binding of the precursor protein to the mitochondrial surface.
- The precursor protein is transferred to the ________, part of the TOM (Translocase of the Outer Membrane) complex.
——- forms a channel that allows the precursor protein to pass through the outer membrane into the intermembrane space.
- The translocating precursor protein reaches a ________, where the outer and inner mitochondrial membranes are closely aligned.
Here, the TIM (Translocase of the Inner Membrane) complex, specifically _________, interacts with the protein.
- The precursor protein is threaded through the ——– translocase, which spans the inner mitochondrial membrane.
_______, an associated protein, helps recruit _________.
ATP hydrolysis by ——– facilitates the stepwise movement of the precursor protein into the matrix.
- Once inside the mitochondrial matrix, the matrix-targeting sequence is removed by a ___________.
This cleavage is necessary to prevent mistargeting and to allow proper folding.
- The imported protein folds into its functional conformation, sometimes assisted by chaperones such as mitochondrial Hsp60.
If necessary, post-translational modifications occur to ensure full activation of the protein.
precursor proteins;
matrix-targeting sequence;
Cytosolic Hsp70 (heat shock protein 70);
import receptors;
Tom40 translocase;
contact site;
Tim23/17;
Tim44;
mitochondrial Hsp70 (mtHsp70);
matrix processing protease
remove the N-terminal signal sequence
signal peptidase
Protein Transport into the Inner Mitochondrial Membrane and the Intermembrane Space Requires Two Signal Sequences
Signal sequence and transmembrane segment
Signal sequence
ATP synthase subunit 9
= Inner membrane (path B)
ADP/ATP antiporter
= Inner membrane (path C)
Cytochrome b2
= Intermembrane space (path A)
Cytochrome c heme lyase
= Intermembrane space (path B)
Porin (P70)
= Outer membrane
1st cleavage by matrix protease
2nd cleavage by protease in intermembrane space (intermembrane space-targeting sequence)
Oxa1
In which membrane does electron transport chain occur
Inner mitochondrial membrane
Intermembrane space
Low pH
High H+ concentration
Matrix
High pH
Low H+ concentration
Mitochondrial Electron Transport Chain
- Electron Donors (NADH & FADH₂)
NADH and FADH₂, generated from the Krebs Cycle, donate electrons to the ETC.
NADH donates electrons to Complex I (NADH dehydrogenase), while FADH₂ donates to Complex II (succinate dehydrogenase).
- Electron Transfer & Proton Pumping
Electrons move through a series of protein complexes (I to IV) in the inner mitochondrial membrane.
As electrons pass through Complex I, III, and IV, protons (H⁺) are pumped from the matrix into the intermembrane space, creating a proton gradient.
- Cytochrome c & Oxygen as the Final Electron Acceptor
Electrons are carried by mobile electron carriers like cytochrome c to Complex IV (cytochrome c oxidase).
At Complex IV, electrons combine with O₂ (oxygen) and protons (H⁺) to form water (H₂O).
Oxygen is the final electron acceptor, making this an aerobic process.
- ATP Synthesis (Oxidative Phosphorylation)
The proton gradient creates a high H⁺ concentration in the intermembrane space and a low H⁺ concentration in the matrix.
Protons flow back into the matrix through ATP synthase (Complex V), driving the conversion of ADP + Pᵢ → ATP.
4 pathways transports from cytosol to inner mitochondrial membrane:
(A) Direct Translocation via TIM23 Complex
Proteins with an N-terminal signal sequence pass through the TOM complex into the TIM23 complex.
If they contain a transmembrane segment, they are inserted into the inner membrane instead of entering the matrix.
(B) TIM22-Mediated Insertion
Proteins that lack an N-terminal signal sequence but have internal targeting signals use intermembrane-space chaperones.
They are recognized by the TIM22 complex, which inserts them into the inner membrane.
(C) OXA-Dependent Insertion of Mitochondrial-Synthesized Proteins
Proteins synthesized inside the mitochondria are inserted into the inner membrane by the OXA complex.
(D) OXA-Dependent Insertion of Imported Proteins
Proteins first enter the matrix via TIM23, where the signal sequence is cleaved.
A secondary signal sequence directs them to the OXA complex, which inserts them into the inner membrane.
Mitochondrial proteins are synthesized in the cytosol or inside the mitochondria and follow distinct pathways for correct localization.
TOM complex serves as the entry gate for cytosolic proteins.
TIM23 is responsible for direct translocation, while TIM22 handles carrier proteins.
OXA complex integrates both mitochondria-synthesized and imported proteins into the inner membrane.
The import pathway used to insert metabolite carrier proteins into the inner mitochondrial membrane utilizes the __________, which is specialized for the translocation of multipass membrane proteins.
TIM22 complex
Recognizes and imports precursor proteins with a chloroplast signal sequence.
Works in conjunction with the TIC complex.
TOC Complex (Translocase of the Outer Chloroplast Membrane)
Facilitates protein translocation across the inner membrane into the stroma.
This step is GTP- or ATP-dependent.
TIC Complex (Translocase of the Inner Chloroplast Membrane)
Once in the stroma, the chloroplast signal sequence is cleaved off, revealing a thylakoid signal sequence for further targeting.
After reaching the stroma, proteins take one of three routes to reach the thylakoid space:
Three Major Pathways for Protein Transport:
- Sec Pathway (ATP + H⁺ electrochemical gradient)
- OXA Pathway (GTP + H⁺ electrochemical gradient)
- Utilizes a signal recognition particle (SRP) homolog for targeting. - TAT Pathway (H⁺ electrochemical gradient)
- Powered only by the proton motive force (H⁺ electrochemical gradient).
Recognizes proteins with a twin-arginine (RR) signal sequence.
Transports fully folded proteins into the thylakoid lumen, making it unique among the three pathways.
Transport in thylakoid space
Proteins destined for the thylakoid space pass through TOC and TIC complexes into the stroma before taking different pathways to the thylakoid.
Transport requires GTP, ATP, or the proton gradient.
Cleavage of chloroplast signal sequence
Cleavage of thylakoid signal sequence
Cleavage of the N-terminal stromal import sequence by a __________ reveals the thylakoid-targeting sequence
stromal protease
___________
* A.k.a. microbodies
* differ from lysosomes in the type of enzyme they hold.
* Peroxisomes hold on to enzymes that require oxygen (________).
Peroxisomes;
oxidative enzymes
Peroxisomes
* contain oxidative enzymes, such as ______ and _______
catalase;
urate oxidase
Peroxisomes
- they are surrounded by only a single membrane, and they do not contain DNA or ribosomes
- all of their proteins must be imported
Anatomy of peroxisome
Lipid bilayer
Crystalline core
Peroxisomes Use ________ and __________ to Perform Oxidative Reactions
Molecular Oxygen;
Hydrogen Peroxide
- Peroxisomes contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from specific organic substrates (designated here as R) in an oxidative reaction that produces hydrogen peroxide(H2O2)
- This type of oxidative reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various toxic molecules that enter the bloodstream
RH2 + O2 -> R + H2O2
2H2O2 -> 2H2O + O2
Peroxisomes function
Involves in β-oxidation (breakdown of very long fatty acid molecules)
a set of reactions that converts glucose to pyruvate or lactate (+2 ATP)
Glycolysis
describes the metabolic shift in cancer cells where they preferentially use glycolysis for energy production, even in the presence of oxygen, resulting in high glucose uptake and lactate production.
Pyruvate -> Lactate
Warburg effect
