CB Chapter 15

What must occur for nuclear import to happen?

• Cargo protein must have correct Nuclear Localization Signal (amino acid sequence) in order to be sent to the nucleus • NLS is recognized and bound by a Nuclear Transport Receptor in cytosol (e.g. importin), which transports cargo protein into the nucleus via pore

What must occur for nuclear export to happen?

• Cargo protein will encounter a small, GTP-bound protein in the nucleus called the Ran protein • The Ran protein causes the release of the cargo protein in the nucleus from the nuclear transport receptor, which it accompanies back across the nuclear membrane to the cytoplasm • The GTP bound to the Ran protein is hydrolyzed causing Ran to be released from the nuclear transport receptor to be recycled

What are the two populations of ribosomes?

1) Free polyribosomes in the cytosol 2) Polyribosomes bound to endoplasmic reticulum membrane by multiple nascent polypeptide chains

What cannot pass through the nuclear pores of the nuclear envelope? What must they carry in order to pass through?

Larger molecules (RNAs & proteins) and macromolecular complexes cannot pass through pores unless they carry the appropriate sorting signal called the nuclear localization signal (NLS)

What are the three major intercellular compartments and what do each include?

1) Endomembrane system: ER, Golgi Apparatus, Lysosomes, Secretory vesicles, Nuclear Envelope, Plasma Membrane 2) Cytosol: Cytoskeleton, Chaperons, Etc. 3) Major organelles: Mitochondria, Chloroplast, Interior of Nucleus

What is the process of receptor-mediated Low Density Lipoproteins (LDL) Endocytosis?

1) LDL is recognized by LDL receptors and they bind and make a coated pit until it is drawn inside the cell in clathrin coated vesicles (it stays here until it fuses with an endosome) 2) Vesicle looses its clathrin coat when it fuses with the endosome; uncoating 3) Uncoated vesicle fuses with an acidic endosome 4) Acidic environment causes LDL to release from its receptors 5) LDL receptors travel back to the plasma membrane via transport vesicles to be reused; recycled 6) The LDL itself is sent to the lysosome where it is further degraded by hydrolytic enzymes and free cholesterol is now made available for various cellular uses

What are the three sorting pathways that a transport vesicle can take in an early endosome?

1) Recycling: The plasma membrane along with those items in and on the membrane (e.g. receptors, channels, pumps, etc.) are returned to the same plasma membrane domain from which they came. 2) Transcytosis: The plasma membrane along with the contents of the transport vesicle fuses with and empties its contents at a different plasma membrane domain than the one from which it came. 3) Degradation: With addition of key enzymes will become lysosomes

What are the two main functions of vesicle coating?

1) Shapes membrane into a bud 2) Helps retain captured molecules

In what three ways do membrane-enclosed organelles import materials?

1) Transport through nuclear pores 2) Transport across membrane 3) Transport by vesicles

What is an antibody composed of and where is it assembled?

Antibody is composed of 4 separate polypeptide chains (2 heavy chains and 2 light chains) and is assembled in the ER

What is protein modification?

Any enzymatically catalyzed modifications of amino acids & enzymatic cleavage or cross linking of peptide

What are the two sides of the golgi apparatus and in what direction are they facing?

Cis- side facing nucleus (entry) Trans- side facing plasma membrane (exit)

What are the two pathways that can occur after protein modification? - What are the polypeptides destined for? - What happens with the ribosomes?

Co-translational modification: polypeptides destined for the endomembrane or for export from the cell; Cytosolic ribosomes become embedded in ER membrane (rough ER) and then finish the translation process The Post-translational: polypeptides destined for the cytosol, mitochondria, chloroplast, peroxisome, or nuclear interior; Translating ribosome remains free in the cytosol

What are the two pathways vesicles can go through once released from the golgi apparatus?

Constitutive secretion and regulated secretion

What does a lysosome contain?

Contains ~40 types of enzymes • All acid hydrolases (i.e. optimal working pH ~5) • Leaked enzymes will not work under cytosolic neutral pH 7

What do disulfide bonds do in the rough ER?

Disulfide (covalent) bonds form to stabilize protein structure. • Stabilize against degradative enzymes found in the cytosol • Stabilize against changes in pH from lumen of ER to cytosol

What does each nuclear pore of the nuclear envelope contain? What does it allow to pass through?

Each pore contains one or more water-filled channels through which small water-soluble molecules can pass freely and non-selectively btw the nucleus and cytosol

What are the differences between the endocytic pathway and the secretory pathway?

Endocytic Endocytosis; bringing in things into the cell via the plasma membrane which form vesicles and pinch off to the inside of the cell to form endosomes which deposit its goods to lysosomes (inward movement) Secretory Exocytosis; moving things out of the cell by forming vesicles in the ER that pinch off and go to the golgi apparatus which release vesicles that either go to the lysosome or travel through the cytosol and exit via the plasma membrane (transport vesicles)

What are endosomes an important part of and why?

Endosomes play an important part in membrane protein maintenance because the list of proteins that are recognized to recycle to the membrane surface has expanded markedly over the past decade

What is used to drive nuclear transport?

Energy supplied by GTP hydrolysis

Why do we see disulfide bond formation in the rough ER?

Enzyme for disulfide bonds are present only in lumen of ER due to reducing environment of the cytosol

Why must there be a balance between endocytosis and exocytosis?

In order for the cell to maintain its original size • Exocytosis would continually enlarge the cell if it weren't balanced with endocytosis • Endocytosis would continually shrink the cell if it weren't balanced with exocytosis

What maintains the acidic conditions within lysosome?

H+ pumps

In endosome recycling, what is returned to the plasma membrane?

In addition to the recycling of important transport receptors (e.g. LDL receptors), there are ion channels, ion pumps, adhesion molecules, and anything else that occurs in or on the plasma membrane that is returned to its original domain

What do protein post-translational modifications (PTMs) do?

Increase the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of entire proteins

What happens when the quality control system of the ER becomes overtaxed or chaperone proteins become overwhelmed and there is a build up of unfolded proteins in the ER? - What does this response cause an increase in production of? - What does physically expand? - What occurs if these two things don't fix the problem?

It trigger Unfolded Protein Response (UPR) ◦ Increase the production of chaperone proteins ◦ Physically expand the ER (prevents ER from becoming clogged with unfolded proteins) ◦ Apoptosis occurs if the first two steps don't work (cell suicide)

Where is the golgi apparatus located?

Located near the nucleus; in animals also close to centrosome

Describe the lumen of an endosome.

Lumen is sub-divided into many compartments by its constantly moving invaginations, which causes its shape to be constantly changing and giving it a transient structure

What happens to late endosome vesicles in endosome degradation?

Many late endosome vesicles become or fuse with an existing lysosome

What are the characteristics of the Nuclear Pore Complex (NPC)?

Massive • Comprised of 456 proteins referred to as nucleoporins • 30 different types of nucleoporins Membrane spanning Inner & Outer ring • Fused to form channel 1/10 micron in diameter

What does the membrane of lysosomes contain?

Membrane contains transporters that allow final products of digestion (e.g. AA, sugars, nucleotides, etc.) to be transferred to cytosol to be reused or excreted from the cell

How is the "sewing effect" produced when polypeptides are trying to enter the ER lumen?

Multiple start and stop transfer sequences within the polypeptide can work in pairs to produce a "Sewing Effect"

What is a lysosome and what is it responsible for?

Membranous sac of hydrolytic enzymes that is responsible for controlled intracellular digestion of both extracellular materials and defective organelles and debris

Nuclear envelope in all eukaryotic cells is perforated by what?

Nuclear pores (~2000)

How does regulated secretion of vesicles released from the golgi apparatus occur? - What does it occur in? - What does it secrete? - What can accumulate and where?

Occurs in special secretory cells • Secretes hormones, mucus, digestive enzymes, etc. • Vesicles can accumulate at plasma membrane awaiting signal to release contents

How does the formation of a disulfide bond occur?

Oxidation of -SH groups of adjacent pairs of cysteine side chains in separate polypeptide chains or on the same chain

What would be an example of regulated vesicle secretion in the golgi apparatus?

Pancreatic beta cell

What are the two main types of endocytosis?

Phagocytosis ("cellular eating") • Ingestion of large particles (microorganisms or cell debris) via large vesicles called phagosomes (>250nm in diameter) • Mainly done by specialized phagocytic cells Pinocytosis ("cellular drinking") • Ingestion of fluid and molecules via small vesicles (<150nm in diameter) • Small amount of material brought in

Phagocytosis in single-cell organisms is for nutrition. In multicellular organisms, what is the process of phagocytosis is used for?

Phagocytosis is used as a defense mechanism—Ingest your Enemies! • Phagocytic white blood cells (e.g. macrophages, neutrophils) defend the body by ingesting invading pathogens like bacteria • Foreign invader recognized by proteins on its surface or chemical signal

Where does polypeptide synthesis begin? What occurs after the translation?

Polypeptide synthesis begins in the cytosol on cytosolic ribosomes, but after the translation of ~30 amino acids the rest of the translation process can take 1 of 2 routes to complete the process called a "modification"

What are the signal sequences of polypeptides based on?

Positively and negatively charged amino acids and hydrophobic amino acids

What needs to occur in order to keep Low Density Lipoproteins (LDL) levels low in the blood?

Receptor-mediated LDL Endocytosis • LDL receptors are found on plasma membrane surfaces to concentrate the uptake of LDL into the cell and out of the blood stream • LDL receptors are searching for and binding to sets of proteins only found on LDL particles

What are two situations that can happen when protein enter the ER lumen?

Situation 1 • Signal sequence almost always at N-terminus • Signal sequence not only directs polypeptide but also opens translocation channel • Chaperons guide the protein through the channel; peptidase cleaves signal sequence; and channel is plugged; and protein remains in the lumen Situation 2 • Note the special sequence farther down the polypeptide called the hydrophobic stop-transfer sequence (not at N-terminus) • Two sequences remain in the channel causing a loop of the polypeptide to enter the ER lumen • The signal peptidase preforms its normal function by cutting the signal sequence from the N-terminus of the polypeptide leaving the C-terminus end in the cytosol and the N-terminus in the lumen of the ER

What is the main duty of an endosome?

Sorting material that enters the cell via endocytosis

How does constitutive secretion of vesicles released from the golgi apparatus occur?

Steady, continuous stream of vesicles from ER to Golgi apparatus (unregulated) • Lipids (plasma membrane): replacement due to endocytosis; prepare for cytokinesis • Proteins: peripheral proteins; diffusion into extra cellular fluid (collagen; elastin)

What is the first step in the formation of glycoproteins in the rough ER?

Step One: Glycosylation • On the cytosolic side of the ER, the addition of a pre-fabricated oligosaccharide (~14 sugars) to a specialized lipid called a dolichol pyrophosphate • The lipid-linked oligosaccharide is flipped with the sugar now being in the lumen of the ER

What is the second step in the formation of glycoproteins in the rough ER?

Step Two: N-linking (most common) • Oligosaccharide protein transferase catalyzes the transfer of the oligosaccharide from the dolichol to the amino group (NH2) of an Asparagine in a polypeptide being translated by ribosomes in the rough ER • Not all asparagine amino acids are involved, only those occurring in a particular tripeptide sequence (e.g. asparagine-X-serine or asparagine-X-threonine)

What occurs in the absence of a sorting signal sequence (default)?

The polypeptide stays in the cytosol

What happens when clathrin triskelia interact with each other?

They form a polyhedral lattice that surrounds the vesicle

What are the two types of proteins that enter the endoplasmic reticulum? What are their characteristics? - How are they translocated? - What are they destined for?

Water-soluble proteins • Completely translocated across the ER membrane and are released into the ER lumen (interior) • Destined to remain and function within the ER lumen or interior of another organelle • Destined for secretion (in vesicle) to golgi apparatus or another location Transmembrane proteins • Partly translocated across the ER membrane and become embedded in membrane • Destined to reside in the ER membrane or membrane of another organelle or plasma membrane • Thus, ER can release sections of membrane already fitted with proteins

What are involved in making a vesicle?

• Cargo: items needed by the cell or disposed of by the cell • Cargo Receptors: proteins that recognize and bind to cargo molecules needing to enter or exit the cell; several types • Adaptin: proteins that bind to loaded cargo receptors on one end and to a clathrin triskelion at the other end to form a vesicle • Dynamin: small GTP-binding protein that assembles as a tightening ring around the neck of the vesicle that pinches off and frees the vesicle from the membrane • Naked Transport Vesicle: Once dynamin frees vesicle adaptin releases the loaded receptor and is recycled along with clathrin; Vesicle is reduced to a cargo surrounded by a membrane embedded with receptors

What do chaperone proteins in the rough ER do?

• Chaperone proteins in the ER will retain defective proteins (e.g. incomplete or incorrectly assembled multimeric proteins; misfolded or truncated proteins) until the protein is corrected • If the protein cannot be corrected, it will be exported for proper disposal

What is vesicle coating and what is it used in?

• Distinct protein coat forms on cytosolic side of membrane only to be shed after bud is pinched off and vesicle is fully formed • Used in secretory and endocytic pathways

What is the function of Binding Immunoglobulin Protein (BiP)?

• Each subunit (polypeptide chain) of the antibody is retained in the ER by this chaperone protein until all the polypeptides are translated and assembled • Nothing is shipped via vesicle until the antibody is assembled

How does vesicle docking occur?

• Each transport vesicle displays a molecular marker (Rab Protein) on its surface that identifies its origin, contents, and destination • Rab is initially recognized by a tethering protein on the cytosolic side of the target membrane. • Tethering protein hold vesicle in place until other proteins called snares, on both the vesicle (v-snare) and target membrane (t-snares) recognize and interact (intertwine) with each other • Vesicle has docked but not fused

What are the two compartments that the endosome is divided into and where are they located?

• Early endosomes located near the periphery of the cell fuse with newly formed endocytic vesicles (pH 6.2-6.5) • Late endosomes located closer to the nucleus and Golgi apparatus and can form lysosomes (pH 5.5)

How does a lysosome cause the terminal ending for endocytosis?

• Endocytic vesicles that contain extracellular fluid and membrane cargo fuse with early endosomes • Sort cargo for recycling, transcytosis or degradation

What are clathrin triskelions and what are they composed of?

• Essentially is the framework outside of the plasma membrane that makes up the vesicle • Composed of three clathrin heavy chains and three light chains

How does vesicle fusion occur?

• For the vesicle to fuse with the target membrane the membranes must be brought within 1.5nm at which time the phospholipids can begin to intermix (i.e. coalesce) • Fusion requires the t-snares and v-snares to continue to interact (i.e. intertwine) • T-snares and V-snares will continue to twist bringing the two membranes closer together, driving out even water molecules until the two membranes fuse • The target membrane will enlarge with every fused vesicle; it will enlarge by the size of the vesicle

How does a lysosome cause the terminal ending for autophagy?

• In autophagy, cytoplasmic material like damaged or surplus organelles is targeted for entrapment by the phagophore, a double-bilayer membrane derived from the ER, forming the autophagosome • Autophagosomes experience a maturation process, ultimately fusing with lysosomes

What is the protein quality job for the golgi apparatus in the retention and retrieval of soluble rough ER proteins?

• KDEL receptors (yellow) found in ER recognize special AA sequences (Lys-Asp-Glu-Leu-COO-) called the ER retention signal in proteins • KDEL receptors are sent to cis side of Golgi apparatus to retrieve proteins with ER retention signals • Once bound together, the KDEL receptor and protein are sent back to ER to be reprocessed

What is the half-life of Low Density Lipoproteins (LDL) receptors and what occurs during this lifespan?

• LDL receptors have a lifespan of ~ 20 hours • During its lifespan, LDL receptors will make one roundtrip from the cell surface to the endosome and back every 10 minutes, whether it is occupied or not • This means a receptor will make hundreds of such trips in its lifetime

How does a lysosome cause the terminal ending for phagocytosis?

• Large extracellular particles (i.e. bacteria) are engulfed by the plasma membrane and sequestered within a phagosome • Phagosomes mature by sequentially fusing with early and late endosomes, and ultimately lysosomes

In endosome degradation, what do late endosomes contain and what do they initiate?

• Late endosomes contain some lysosomal enzymes, so digestion of cargo proteins begins in the late endosome and continues as the endosome gradually matures into a lysosome • Once the late endosome has digested its contents, it takes on a dense, rounded appearance which is characteristic of a classical lysosome

What do Low Density Lipoproteins (LDL) do and what happens if levels remain high in the blood?

• Low Density Lipoproteins (LDL) transport cholesterol from the liver to the tissues of the body • If high levels remain in the blood, LDL contributes to build up of plaque in the arteries and subsequent, heart disease

What is the pathway of disposal for misshaped proteins that cannot be fixed in the ER?

• Misfolded glycosylated proteins are escorted out of ER by chaperone proteins via protein translocator • Enzyme in the cytosol, N-glycanase, removes polysaccharide from misfolded protein • Ubiquitin escorts misfolded protein to a proteasome that breaks down proteins into pieces of which can be recycled (protein garbage disposal)

What does receptor-mediated endocytosis provide and help limit? What is the best example of this?

• Provides a selective concentrating mechanism that increases the efficiency of endocytosing the correct molecules by 1000-fold • This same mechanism helps limit the extra-cellular fluid taken in during endocytosis • Best example: Endocytosis of cholesterol (Cholesterol is the extremely insoluble lipid used to stabilize membranes)

What happens to proteins that are destined to the ER?

• Signal Recognition Particle (SRP) recognizes the ER signal sequence brings the ribosome with the polypeptide that is in the process of translating to the SRP receptor • Once bound, it brings the translating ribosome to the ER and diffuse until it finds a translocation channel • Once it does, the SRP is displaced and recycled • Ribosome continues to translate the polypeptide and at least half of polypeptide will be in the ER lumen

What are the major players involved in proteins entering the mitochondria and chloroplast?

• Signal Sequence: specific amino acid sequence that determines that a certain protein belongs in the mitochondria or the chloroplast • Receptor Protein: on the outer membrane that looks for the specific signal sequence and once bound, the receptor and protein will diffuse across the outer membrane until it encounters the contact site • Contact Site: includes protein translocators that allow the protein to enter the matrix • Chaperone Proteins: responsible for the unfolding and folding of proteins once it enters the matrix • Signal Peptidase: cleaves signal sequence to form a mature protein

How do proteins travel through the golgi apparatus?

• Soluble proteins travel through the Golgi apparatus (cis to trans) via a transport vesicle originating from the ER and fusing with one cisterna to the next • Proteins can go through further modifications in the Golgi apparatus such as a more complex oligosaccharide chain can be added

How is there a formation of a lysosome?

• The specialized digestive enzymes and membrane proteins of the lysosome are synthesized in the ER and transported through the Golgi apparatus to the cis side • The enzymes have been tagged with a specific phosphorylated sugar group (mannose 6-phosphate) that ensures such enzymes will be delivered to the lumen of the lysosome • The lumen side of the lysosome membrane is heavily glycosylated which prevents the membrane proteins from being digested by the lysosome proteases

What does the golgi apparatus contain?

◦ Collection of flattened, membrane-enclosed sacs (lumen) called cisternae (Average stack can contain 3-20 cisternae) ◦ Depending of cell type: one large cisterna to hundreds cisternae per stack