Using the following powerpoint answer the followingquestions on the study guide.
THESE ARE THE QUESTIONS:
1.)Be able to explain why ER signal sequences are thought to benecessary and sufficient
2.)Know how the cell regulates the activity of transporters,receptors, and enzymatic proteins.
3.)Be able to explain, in moderate detail, the three mainmechanisms of protein transport into organelles.
4.)Be able to describe the transport of soluble, single-pass anddouble-pass transmembrane proteins across the ER membrane.
5.)Know what happens to improperly folded and incompletelymodified proteins.
6.)Be able to explain how the different types of ion channelsare used by neurons to receive and transmit information.
7.)Be able to explain the formation of clathrin-coatedvesicles
CHAPTER CONTENTS
MEMBRANE-ENCLOSED ORGANELLES
PROTEIN SORTING
VESICULAR TRANSPORT
SECRETORY PATHWAYS
ENDOCYTIC PATHWAYS
MEMBRANE-ENCLOSED ORGANELLES
Eukaryotic Cells Contain a Basic Set of Membrane-enclosedOrganelles
PROTEIN SORTING
Proteins Are Transported into Organelles by Three Mechanisms
Signal Sequences Direct Proteins to the Correct Compartment
Proteins Enter the Nucleus Through Nuclear Pores
Proteins Unfold to Enter Mitochondria and Chloroplasts
Proteins Enter Peroxisomes from Both the Cytosol and theEndoplasmic Reticulum
Protein Sorting
Proteins are made in the cytoplasm (by ribosomes either free inthe cytosol or on the rough ER).
Proteins must then be transported either into the RER lumen, orto another site in the cell.
How are these new proteins sorted?
Protein Sorting
Cytosol
Nucleus
Mitochondria and Chloroplasts
Endoplasmic Reticulum
Soluble proteins (lumen)
Membrane proteins
3 main mechanisms for importing proteins into membrane-boundorganelles
PROTEIN SORTING
Proteins Are Transported into Organelles by Three Mechanisms
Signal Sequences Direct Proteins to the Correct Compartment
Proteins Enter the Nucleus Through Nuclear Pores
Proteins Unfold to Enter Mitochondria and Chloroplasts
Proteins Enter Peroxisomes from Both the Cytosol and theEndoplasmic Reticulum
Protein Signal Sequences
Signal sequences are a stretch of amino acids typically 15-60amino acids long found on the N-terminal of a newly synthesizedprotein
They are often (but not always) removed from the finishedprotein once they have been sorted correctly.
Signal sequences allow the newly synthesized proteins to berecognized and delivered to the proper place.
The exact sequence of the signal sequence does not seem to be asimportant as its physical propeties (hydrophobicity, placement ofcharged amino acids…)
Signal sequences are necessary and sufficient for proteinsorting.
Proteins without a signal sequence stay in the cytosol
Protein Signal Sequences
Table 15–3 Some Typical Signal Sequences
FUNCTION OF SIGNAL EXAMPLE OF SIGNAL SEQUENCE
Import into ER +H3N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe- Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-
Retention in lumen of ER -Lys-Asp-Glu-Leu-COO–
Import into mitochondria +H3N-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-
Import into nucleus -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-
Import into peroxisomes -Ser-Lys-Leu-
PROTEIN SORTING
Proteins Are Transported into Organelles by Three Mechanisms
Signal Sequences Direct Proteins to the Correct Compartment
Proteins Enter the Nucleus Through Nuclear Pores
Proteins Unfold to Enter Mitochondria and Chloroplasts
Proteins Enter Peroxisomes from Both the Cytosol and theEndoplasmic Reticulum
PROTEIN SORTING
Proteins Are Transported into Organelles by Three Mechanisms
Signal Sequences Direct Proteins to the Correct Compartment
Proteins Enter the Nucleus Through Nuclear Pores
Proteins Unfold to Enter Mitochondria and Chloroplasts
Proteins Enter Peroxisomes from Both the Cytosol and theEndoplasmic Reticulum
PROTEIN SORTING
Proteins Are Transported into Organelles by Three Mechanisms
Signal Sequences Direct Proteins to the Correct Compartment
Proteins Enter the Nucleus Through Nuclear Pores
Proteins Unfold to Enter Mitochondria and Chloroplasts
Proteins Enter Peroxisomes from Both the Cytosol and theEndoplasmic Reticulum
Import into the Peroxisomes
Enzymes that break down toxins, fatty acids and alcohol areimported into peroxisomes.
Imported using three a.a. signal sequence, receptor proteins andprotein translocators (similar to mitochondrial proteins).
The proteins do not unfold first
Exact mechanism still not clear.
PROTEIN SORTING
Proteins Enter the Endoplasmic Reticulum While BeingSynthesized
Soluble Proteins Made on the ER Are Released into the ERLumen
Start and Stop Signals Determine the Arrangement of aTransmembrane Protein in the Lipid Bilayer
Import into the ER
Two types of proteins are imported into the ER:
Water-soluble proteins
These are destined for either secretionor for the lumen of an organelle
Prospective trans-membraneproteins
These are destined to reside in themembrane of the ER or other cellular organelle or the Plasmamembrane
Import into the ER
Unlike Nuclear, Mitochondrial and Chloroplast proteins, most ofthe ER proteins are threaded into the ER while beingtranslated.
Membrane-bound ribosomes are located on the surface of the roughER and make proteins that are being translocated across the ERmembrane
Free ribosomes not located on any membranes make all otherproteins
PROTEIN SORTING
Proteins Enter the Endoplasmic Reticulum While BeingSynthesized
Soluble Proteins Made on the ER Are Released into the ERLumen
Start and Stop Signals Determine the Arrangement of aTransmembrane Protein in the Lipid Bilayer
Import into the ER
While the growing polypeptide chain is being produced, asignal-recognition particle (SRP) binds to the ER signalsequence.
The SRP is then recognized by an SRP receptor on the surface ofthe ER.
The SRP dissassociates and the SRP receptor brings the ribosometo a translocation channel where the new polypeptide enters the ERlumen while it is being translated.
Once inside the ER lumen, a signal peptidase cleaves off thesignal sequence
PROTEIN SORTING
Proteins Enter the Endoplasmic Reticulum While BeingSynthesized
Soluble Proteins Made on the ER Are Released into the ERLumen
Start and Stop Signals Determine the Arrangement of aTransmembrane Protein in the Lipid Bilayer
Transmembrane Protein import
Remain embeded in membrane of ER – not released to the lumen
Single membrane spanning proteins contain a sequence ofhydrophobic amino acids called a stop-transfer sequence that causesthe protein to be released by the translocation channel andinserted into the membrane.
N-terminal remains in lumen, C-terminal in cytosol
15_15_into_ER_membr.jpg
Transmembrane Protein import
Multiple membrane spanning proteins have an internal signalsequence (not an N-terminal sequence) that doubles as astart-transfer sequence.
When the channel recognizes the start-transfer sequence, itcauses the protein to translocate in the other direction across themembrane. Hydrophobic a-helices span the membrane until astop-transfer sequence is recognized.
15_16_double_pass.jpg
Vesicular Transport
Entry into ER often only first step to final destination
Initial destination after ER is Golgi complex
Transport from ER to Golgi, between Golgi stacks, and from Golgito either lysosomes or cell surface carried out by transportvesicles
Transport vesicles continually bud off from one compartment andfuse to another
Transport can occur in forward and reverse directions
Vesicles are specific for the distinct proteins and lipids theycarry
VESICULAR TRANSPORT
Transport Vesicles Carry Soluble Proteins and Membrane BetweenCompartments
Vesicle Budding Is Driven by the Assembly of a Protein Coat
Vesicle Docking Depends on Tethers and SNAREs
VESICULAR TRANSPORT
Transport Vesicles Carry Soluble Proteins and Membrane BetweenCompartments
Vesicle Budding Is Driven by the Assembly of a Protein Coat
Vesicle Docking Depends on Tethers and SNAREs
15_18_Clathrin_EM.jpg
Clathrin-Coated Vesicles
Clathrin-coated vesicles are the best studied
Involved in both the outward secretory and inward endocytoticpathways.
Adaptins secure the clathrin coat to the vesicle membrane andhelp select the cargo molecules for transport.
Adaptins trap cargo receptors that bind to the cargomolecules.
Dynamin molecules bind to GTP (energy carrier) and pinch off thecell membrane into a vesicle.
15_19_Clathrin_vesicle.jpg
VESICULAR TRANSPORT
Transport Vesicles Carry Soluble Proteins and Membrane BetweenCompartments
Vesicle Budding Is Driven by the Assembly of a Protein Coat
Vesicle Docking Depends on Tethers and SNAREs
Vesicular transport
Coated vesicles are transported along fibers of the cytoskeletonto their final destination.
The vesicle recognizes and docked with its correct organellethrough a family of transmembrane proteins called SNAREs.
v-SNAREs are on the transport vesicles and their complementaryt-SNAREs are found on the target membrane of the organelle.
Each organelle and each type of transport vesicle is thought tocontain unique SNAREs.
Once the SNAREs recognize each other, the vesicle docks with theorganelle and membrane fusion occurs.
15_20_SNAREs.jpg
SECRETORY PATHWAYS
Most Proteins Are Covalently Modified in the ER
Exit from the ER Is Controlled to Ensure Protein Quality
The Size of the ER Is Controlled by the Demand for Protein
Proteins Are Further Modified and Sorted in the GolgiApparatus
Secretory Proteins Are Released from the Cell by Exocytosis
Secretory Pathways
Exocytosis is the process by which newly made proteins, lipidsand carbohydrates are delivered to the cell surface by transportvesicles which fuse with the cell membrane.
Proteins are first chemically modified in the ER – disulfidebonds are formed and glycosylation occurs (carbohydrates arecovalently added to an Asparagine nitrogen (N-linked))
15_22_glycosylated_ER.jpg
SECRETORY PATHWAYS
Most Proteins Are Covalently Modified in the ER
Exit from the ER Is Controlled to Ensure Protein Quality
The Size of the ER Is Controlled by the Demand for Protein
Proteins Are Further Modified and Sorted in the GolgiApparatus
Secretory Proteins Are Released from the Cell by Exocytosis
Secretory Pathways
Proteins without an ER retention signal (on the C-terminus) arepackaged into transport vesicles and sent to the Golgi.
Improperly folded or incompletely modified proteins are retainedby chaperone proteins in the ER and degraded.
Cystic Fibrosis is caused by amutation that leads to misfolding of a chloride channel that leadsto its not being exported
SECRETORY PATHWAYS
Most Proteins Are Covalently Modified in the ER
Exit from the ER Is Controlled to Ensure Protein Quality
The Size of the ER Is Controlled by the Demand for Protein
Proteins Are Further Modified and Sorted in the GolgiApparatus
Secretory Proteins Are Released from the Cell by Exocytosis
Secretory Pathways
An accumulation of misfolded proteins in the ER triggers theunfolded protein response (UPR).
The UPR stimulates increased production of ER chaperone proteinsand increases the size of the ER.
SECRETORY PATHWAYS
Most Proteins Are Covalently Modified in the ER
Exit from the ER Is Controlled to Ensure Protein Quality
The Size of the ER Is Controlled by the Demand for Protein
Proteins Are Further Modified and Sorted in the GolgiApparatus
Secretory Proteins Are Released from the Cell by Exocytosis
Golgi Apparatus
The Golgi Apparatus sorts and further modifies proteins.
After traveling through the Golgi stacks, proteins are packedinto transport vesicles and sent to the cell membrane.
SECRETORY PATHWAYS
Most Proteins Are Covalently Modified in the ER
Exit from the ER Is Controlled to Ensure Protein Quality
The Size of the ER Is Controlled by the Demand for Protein
Proteins Are Further Modified and Sorted in the GolgiApparatus
Secretory Proteins Are Released from the Cell by Exocytosis
Exocytosis and Endocytosis
Exocytosis - mechanism for exporting proteins and lipids out ofthe cell
Endocytosis - mechanism for importing molecules into thecell
Two secretory pathways in secretory cells
All cells contain a constitutive secretory pathway fordelivering plasma membrane proteins and lipid to plasmamembrane.
Also used for secretion of some proteins into blood e.g. albuminfrom liver cells
Specialized cells like hormone producing cells also contain aregulated secretory pathway where secretory vesicles are stored atthe cell membrane and released all at once.
This allows controlled secretion of large quantities of proteinin response to specific stimuli
15_28_trans_Golgi_net.jpg
15_29_Secretory_vesicl.jpg
Endocytic pathways
Pinocytosis (“cellular drinkingâ€) - uptake of fluid andmolecules via small (<150nm diameter) vesicles. Occurs in allcells
Phagocytosis (“cellular eatingâ€) - uptake of large particlese.g. microorganisms and cell debris via large (>250nm diameter)vesicle. Only occurs in specialised cells - phagocytic cells e.g.macrophages
ENDOCYTIC PATHWAYS
Specialized Phagocytic Cells Ingest Large Particles
Fluid and Macromolecules Are Taken Up by Pinocytosis
Receptor-mediated Endocytosis Provides a Specific Route intoAnimal Cells
Endocytosed Macromolecules Are Sorted in Endosomes
Lysosomes Are the Principal Sites of Intracellular Digestion
White blood cell ingests bacteria
Macrophage engulfs red blood cells
ENDOCYTIC PATHWAYS
Specialized Phagocytic Cells Ingest Large Particles
Fluid and Macromolecules Are Taken Up by Pinocytosis
Receptor-mediated Endocytosis Provides a Specific Route intoAnimal Cells
Endocytosed Macromolecules Are Sorted in Endosomes
Lysosomes Are the Principal Sites of Intracellular Digestion
ENDOCYTIC PATHWAYS
Specialized Phagocytic Cells Ingest Large Particles
Fluid and Macromolecules Are Taken Up by Pinocytosis
Receptor-mediated Endocytosis Provides a Specific Route intoAnimal Cells
Endocytosed Macromolecules Are Sorted in Endosomes
Lysosomes Are the Principal Sites of Intracellular Digestion
Receptor-mediated endocytosis
Pinocytosis is indescriminate - cells must also have a way tointernalize selectively
Selected macromolecules are taken up by cell via specificinteraction with a receptor on the cell surface - receptor mediatedendocytosis
Clathrin-coated vesicles are involved
Examples are:
Cholesterol transported in bloodcomplexed to protein - low density lipoprotein (LDL) which binds toreceptors on cell surface and is internalized. Within endosomes LDLand receptor dissociate, LDL transferred to lysosome, degraded andcholesterol released into cytosol.
Other examples include insulin andother signaling hormones, iron and vitamin B12.
ENDOCYTIC PATHWAYS
Specialized Phagocytic Cells Ingest Large Particles
Fluid and Macromolecules Are Taken Up by Pinocytosis
Receptor-mediated Endocytosis Provides a Specific Route intoAnimal Cells
Endocytosed Macromolecules Are Sorted in Endosomes
Lysosomes Are the Principal Sites of Intracellular Digestion
Possible fates for proteins
after endocytosis
Most receptors specifically retrieved from endosomes to samearea of plasma membrane – recycling
If not retrieved, receptors follow pathway from endosomes tolysosomes for degradation
Some receptors are returned to a different area of plasmamembrane - transcytosis. This transports cargo molecules from oneextracellular space to another
