Question

Using the following powerpoint answer the following questions on the study guide.

THESE ARE THE QUESTIONS:

1.)Be able to explain why ER signal sequences are thought to be necessary 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 main mechanisms of protein transport into organelles.

4.)Be able to describe the transport of soluble, single-pass and double-pass transmembrane proteins across the ER membrane.

5.)Know what happens to improperly folded and incompletely modified proteins.

6.)Be able to explain how the different types of ion channels are used by neurons to receive and transmit information.

7.)Be able to explain the formation of clathrin-coated vesicles

CHAPTER CONTENTS

MEMBRANE-ENCLOSED ORGANELLES

PROTEIN SORTING

VESICULAR TRANSPORT

SECRETORY PATHWAYS

ENDOCYTIC PATHWAYS

MEMBRANE-ENCLOSED ORGANELLES

Eukaryotic Cells Contain a Basic Set of Membrane-enclosed Organelles

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 the Endoplasmic Reticulum

Protein Sorting

Proteins are made in the cytoplasm (by ribosomes either free in the cytosol or on the rough ER).

Proteins must then be transported either into the RER lumen, or to 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-bound organelles

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 the Endoplasmic Reticulum

Protein Signal Sequences

Signal sequences are a stretch of amino acids typically 15-60 amino acids long found on the N-terminal of a newly synthesized protein

They are often (but not always) removed from the finished protein once they have been sorted correctly.

Signal sequences allow the newly synthesized proteins to be recognized and delivered to the proper place.

The exact sequence of the signal sequence does not seem to be as important as its physical propeties (hydrophobicity, placement of charged amino acids…)

Signal sequences are necessary and sufficient for protein sorting.

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 the Endoplasmic 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 the Endoplasmic 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 the Endoplasmic Reticulum

Import into the Peroxisomes

Enzymes that break down toxins, fatty acids and alcohol are imported into peroxisomes.

Imported using three a.a. signal sequence, receptor proteins and protein translocators (similar to mitochondrial proteins).

The proteins do not unfold first

Exact mechanism still not clear.

PROTEIN SORTING

Proteins Enter the Endoplasmic Reticulum While Being Synthesized

Soluble Proteins Made on the ER Are Released into the ER Lumen

Start and Stop Signals Determine the Arrangement of a Transmembrane 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 secretion or for the lumen of an organelle

Prospective trans-membrane proteins

These are destined to reside in the membrane of the ER or other cellular organelle or the Plasma membrane

Import into the ER

Unlike Nuclear, Mitochondrial and Chloroplast proteins, most of the ER proteins are threaded into the ER while being translated.

Membrane-bound ribosomes are located on the surface of the rough ER and make proteins that are being translocated across the ER membrane

Free ribosomes not located on any membranes make all other proteins

PROTEIN SORTING

Proteins Enter the Endoplasmic Reticulum While Being Synthesized

Soluble Proteins Made on the ER Are Released into the ER Lumen

Start and Stop Signals Determine the Arrangement of a Transmembrane Protein in the Lipid Bilayer

Import into the ER

While the growing polypeptide chain is being produced, a signal-recognition particle (SRP) binds to the ER signal sequence.

The SRP is then recognized by an SRP receptor on the surface of the ER.

The SRP dissassociates and the SRP receptor brings the ribosome to a translocation channel where the new polypeptide enters the ER lumen while it is being translated.

Once inside the ER lumen, a signal peptidase cleaves off the signal sequence

PROTEIN SORTING

Proteins Enter the Endoplasmic Reticulum While Being Synthesized

Soluble Proteins Made on the ER Are Released into the ER Lumen

Start and Stop Signals Determine the Arrangement of a Transmembrane 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 of hydrophobic amino acids called a stop-transfer sequence that causes the protein to be released by the translocation channel and inserted 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 signal sequence (not an N-terminal sequence) that doubles as a start-transfer sequence.

When the channel recognizes the start-transfer sequence, it causes the protein to translocate in the other direction across the membrane. Hydrophobic a-helices span the membrane until a stop-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 Golgi to either lysosomes or cell surface carried out by transport vesicles

Transport vesicles continually bud off from one compartment and fuse to another

Transport can occur in forward and reverse directions

Vesicles are specific for the distinct proteins and lipids they carry

VESICULAR TRANSPORT

Transport Vesicles Carry Soluble Proteins and Membrane Between Compartments

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 Between Compartments

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 endocytotic pathways.

Adaptins secure the clathrin coat to the vesicle membrane and help select the cargo molecules for transport.

Adaptins trap cargo receptors that bind to the cargo molecules.

Dynamin molecules bind to GTP (energy carrier) and pinch off the cell membrane into a vesicle.

15_19_Clathrin_vesicle.jpg

VESICULAR TRANSPORT

Transport Vesicles Carry Soluble Proteins and Membrane Between Compartments

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 cytoskeleton to their final destination.

The vesicle recognizes and docked with its correct organelle through a family of transmembrane proteins called SNAREs.

v-SNAREs are on the transport vesicles and their complementary t-SNAREs are found on the target membrane of the organelle.

Each organelle and each type of transport vesicle is thought to contain unique SNAREs.

Once the SNAREs recognize each other, the vesicle docks with the organelle 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 Golgi Apparatus

Secretory Proteins Are Released from the Cell by Exocytosis

Secretory Pathways

Exocytosis is the process by which newly made proteins, lipids and carbohydrates are delivered to the cell surface by transport vesicles which fuse with the cell membrane.

Proteins are first chemically modified in the ER – disulfide bonds are formed and glycosylation occurs (carbohydrates are covalently 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 Golgi Apparatus

Secretory Proteins Are Released from the Cell by Exocytosis

Secretory Pathways

Proteins without an ER retention signal (on the C-terminus) are packaged into transport vesicles and sent to the Golgi.

Improperly folded or incompletely modified proteins are retained by chaperone proteins in the ER and degraded.

Cystic Fibrosis is caused by a mutation that leads to misfolding of a chloride channel that leads to 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 Golgi Apparatus

Secretory Proteins Are Released from the Cell by Exocytosis

Secretory Pathways

An accumulation of misfolded proteins in the ER triggers the unfolded protein response (UPR).

The UPR stimulates increased production of ER chaperone proteins and 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 Golgi Apparatus

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 packed into 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 Golgi Apparatus

Secretory Proteins Are Released from the Cell by Exocytosis

Exocytosis and Endocytosis

Exocytosis - mechanism for exporting proteins and lipids out of the cell

Endocytosis - mechanism for importing molecules into the cell

Two secretory pathways in secretory cells

All cells contain a constitutive secretory pathway for delivering plasma membrane proteins and lipid to plasma membrane.

Also used for secretion of some proteins into blood e.g. albumin from liver cells

Specialized cells like hormone producing cells also contain a regulated secretory pathway where secretory vesicles are stored at the cell membrane and released all at once.

This allows controlled secretion of large quantities of protein in response to specific stimuli

15_28_trans_Golgi_net.jpg

15_29_Secretory_vesicl.jpg

Endocytic pathways

Pinocytosis (“cellular drinking”) - uptake of fluid and molecules via small (<150nm diameter) vesicles. Occurs in all cells

Phagocytosis (“cellular eating”) - uptake of large particles e.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 into Animal 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 into Animal 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 into Animal 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 to internalize selectively

Selected macromolecules are taken up by cell via specific interaction with a receptor on the cell surface - receptor mediated endocytosis

Clathrin-coated vesicles are involved

Examples are:

Cholesterol transported in blood complexed to protein - low density lipoprotein (LDL) which binds to receptors on cell surface and is internalized. Within endosomes LDL and receptor dissociate, LDL transferred to lysosome, degraded and cholesterol released into cytosol.

Other examples include insulin and other 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 into Animal 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 same area of plasma membrane – recycling

If not retrieved, receptors follow pathway from endosomes to lysosomes for degradation

Some receptors are returned to a different area of plasma membrane - transcytosis. This transports cargo molecules from one extracellular space to another

Answer 1

1.)Be able to explain why ER signal sequences are thought to be necessary and sufficient.

Signal sequences are necessary for the proteins to be sorted and delivered to their right place, without these signal sequences proteins may stay and end-up in the cytosol, not being able to complete their function. They are sufficient, as the signal sequence is the only thing they need to be delivered to their right place. These signal sequences are usually made of around 15-60 amino acids, located in the N-terminal side of a new synthesized protein.

3.)Be able to explain, in moderate detail, the three main mechanisms of protein transport into organelles.

Proteins are transported into organelles by three main mechanisms: As we mentioned, signal sequences determine and deliver proteins to their right place; once they are directed to their objective:

1. Proteins may enter an organelle through pores, such as in the nucleus, where proteins use the nuclear pores (as passages) to enter the nucleus.
2. Proteins may enter peroxisomes using a 3 amino acid signal sequences (located in the C-terminal side), which is recognized by a receptor protein and then translocated into the peroxisome by a protein translocator. For this process, the protein must NOT unfold. Exact mechanism is not fully understood.
3. Proteins enter the mitochondria through a receptor located on the external membrane, called "Translocase of Outer Membrane", which binds and translocate the protein inside the intermembrane space, where is helped by chaperones to reach the "Translocase of Inner Membrane" to be translocated to the mitochondrial matrix. Proteins unfold in order to enter the mitochondria.

When proteins objective is to stay in the ER, they enter the ER as they are being translated and synthesized.

4.)Be able to describe the transport of soluble, single-pass and double-pass transmembrane proteins across the ER membrane.

Single membrane spanning proteins contain a sequence of hydrophobic amino acids called a stop-transfer sequence that causes the protein to be released by the translocation channel and inserted into the membrane.
N-terminal remains in lumen, C-terminal in cytosol.

Multiple membrane spanning proteins have an internal signal sequence (not an N-terminal sequence) that doubles as a start-transfer sequence. When the channel recognizes the start-transfer sequence, it causes the protein to translocate in the other direction across the membrane. Hydrophobic a-helices span the membrane until a stop-transfer sequence is recognized.

5.)Know what happens to improperly folded and incompletely modified proteins.

Unfolded, improperly folded and incompletely modified proteins, remain in the ER with its chaperones in order to be degraded later on. If a lot of these proteins accumulates, an ER response is triggered: Unfolded Protein Respones (UPR) which stimulates the production of chaperone proteins in order to increase their number and therefore, the degradation of these unfolded proteins. ER sizes increases along with the amount of chaperon proteins being produced by this response.

7.)Be able to explain the formation of clathrin-coated vesicles.

Clathrin-coted vesicles are the main factor involved in vesicular transportation. These vesicles bud from the cell membrane, where the clathrin (a protein complexed made of three heavy chains and three light chains) forms a web or lattice structure needed for the vesicle formation. However, clathrin itself cannot bind to the cell membrane, therefore, this connections is mediated by clathrin adaptors: adaptins, which secures the clathrin scaffolf to the vesicle membrane and helps select the cargo to transport.
Adaptins trap cargo receptors that bind to the cargo molecules.
Dynamin molecules bind to GTP (energy carrier) and pinch off the cell membrane into a vesicle.