Cell & Molecular Biology, Bio 3050, Petcoff, Exam 4

Describe the various modes of cellular digestion and give examples of each (see question 12-6)

*Lysosomes involved in: phagocytosis, receptor-mediated endo, autophagy and extracellular digestion. -Receptor mediated endocytosis. -Phagocytosis: specific form of endocytosis involving the vascular internalization of solids such as bacteria by an organism, and is therefore distinct from other forms of endocytosis such as the vesicular internalization of various liquids (pinocytosis). Phagocytosis is involved in the acquisition of nutrients for some cells. Fuses with late endosome or matures directly to lysosome. Ex: neutrophils and macrophages use for defense. Can involve exocytosis, digested material from extracellular origin, involves acid hydrolases, occurs in lysosomes, serves as source of nutrients in cells. -Autophagy - fusion of vesicles/vacuoles with lysosomes, digested from intracellular origin. Can involve exocytosis, involves acid hydrolases, occurs in lysosomes, serves as source of nutrients in cells. -Extracellular digestion - essential for sperm. Fusion lysosomes with plasma membrane, involve acid hydrolases, digested material extracellular origin, involves exocytosis.

What is the role of clathrin within a cell? Describe how it is used

-Clathrin from latin word lattice. -Unique shape of the clathrin proteins and they way they assemble to form clathrin coats provides the driving force that causes flat membranes to curve and from spherical vesicles.

Describe the role of an ER signal sequence on a polypeptide

-Directs ribosome-mRNA-polypeptide complex to surface of RER. -Typically 15-30 AA's long and have 3 domains: + charged N terminal region, central hydrophobic region, polar region adjoining site where cleavage from mature protein will occur.

Compare and contrast protein trafficking associated withe endocytosis and exocytosis

-Exocytosis, regulated secretion: Ca+ often appears to be essential step in signal cascade leading from receptor on cell surface to exocytosis. Secretory granules. -Most endocytosic vesicles develop into early endosomes which fuse with vesicles from TGN acquiring digestive enzymes and maturing to form new lysosomes. -Receptor mediated endocytosis: Clathrin dependent. Cells use specific receptors found on outer surface of plasma membrane. Ex: LDL internalization. Binding specific ligands to receptors, diffuses into membrane, coated pits that serve as sites fo collection and internalization. Increased receptor-ligand in coated pits triggers accumulation of proteins inner (cytosolic) surface plasma membrane. Adaptor proteins, clathrin, dynamin. Promote membrane curvature and invagination of pit. Pit pinches off from PM forming coated vesicle. Clathrin coats released, free to fuse with early endosome

Diagram and explain how proteins on the inner membrane of the rough ER become part of the outer leaflet of the plasma membrane (and see problem 12-3)

-Glycoproteins always found on outside of phospholipid monolayer with CHO chains exposed because this is the monolayer that originally faced the interior of the RER and Golgi, where enzymes for glycolsylation are. Membrane asymmetry maintained.

Give an example of the functional relationship between peroxisomes and mitochondria in animal cells (hint: it involves lipid catabolism)

-In animal cells, peroxisomal beta oxidation is especially important for degrading long-chain, very long-chain, and branched fatty acids. The primary product of beta oxidation, acetyl-CoA, is then exported to the cytosol. Once fatty acids are shortened to fewer than 16 carbons, further oxidation usually occurs in the mitochondria. -Thus, in animal cells, the peroxisome is important for shortening fatty acids in preparation for subsequent metabolism in the mitochondrion rather than completely breaking them down to acetyl-CoA

• Describe the structure, composition, and function of a lysosome

-Lysosome is an organelle of endomembrane system that contains digestive enzymes, hydrolytic enzymes. Develop from endosomes: synthesized by ribosomes on RER, translocated through pore of ER membrane into lumen before transport to Golgi. Sorted in TGN and packaged in clathrin coats, M6P tag added. Lose clathrin coat and travel to endosome (delivered to endosomal transport vesicles). Early endosome matures to late endosome. Late endosome a collection of newly synthesized digestive enzymes and extracellular/intracellular material fated for digestion. Final step is activation of acid hydrolases, H+ pump drops pH of late endosome thereby transforming late endosome into a lysosome. Late endosome can also transfer material to acid lumen of existing lysosomes. -Lysosomes may release enzymes to outside by exocytosis. Extracellular digestion. -Most important function is degradation of foreign material brought into cell by phagocytosis and receptor-mediated endocytosis. Phagocytic vesicles are transformed into lysosomes by fusing with early endosomes, vesicles with material brought in by receptor-mediated endo also form early endosomes. Early endosomes fuse with vesicles from TGN containing acid hydrolases and mature to late endosomes. -Lysosomes also involved in autophagy - digestion of old and unwanted cell structures/organelles.

How and where do proteins become glycosylated?

-Some glycosylation reactions occur in the lumen of the ER; others, in the lumina of the cis-, medial-, ortrans-Golgi cisternae. Thus the presence of certain carbohydrate residues on proteins provide useful markers for following their movement from the ER and through the Golgi cisternae. -N Glycosylation or pro in ER: Synthesis of core oligosaccharide begins in cytoplasm, dolichol phosphate as carrier. Partially synthesized oligosaccharide is translocated to ER lumen where additional monosaccharides are added. Completed oligosaccharide transferred to target protein, several monosaccharides are removed in final processing. *Inside ER becomes outside of plasma membrane. -Compartmentalization of steps of glycocylation and subsequent modifications of proteins. Terminal gylcosylation in Golgi. Glucan synthase and glycosyl transferases.

What is a zymogen granule and why go about doing things this way?

-Specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. -Type of mature regulated secretory vesicle that contains increased concentration of protein, located in space btwn Golgi and PM bordering lumen into which contents released. -Promote formation of large protein aggregates. Made in grandular cells that secrete enzymes. -Endocrine pancreas: "within", secreted in blood, insulin and glucagon. Exocrine pancreas, acinar cells, digestive enzymes go into small intestine.

Describe the structure of a Golgi apparatus. What is the function of the Golgi? How many of these are found in a cell?

-The Golgi apparatus packages proteins into membrane-bound vesicles inside the cell before the vesicles are sent to their destination. -A series of compartments consisting of two main networks: the cis Golgi network (CGN) and the trans Golgi network (TGN). The CGN is a collection of fused, flattened membrane-enclosed disks known as cisternae (cisterna), originating from vesicular clusters that bud off the ER. A mammalian cell typically contains 40 to 100 stacks. This collection of cisternae is broken down into cis, medial, and trans compartments. The TGN is the final cisternal structure, from which proteins are packaged into vesicles destined to lysosomes, secretory vesicles, or the cell surface. The TGN is usually positioned adjacent to the stacks of the Golgi apparatus, but can also be separate from the stacks. -The number of 'Golgi apparatus' within a cell is variable. Animal cells tend to have fewer and larger Golgi apparatus. Plant cells can contain as many as several hundred smaller versions.

What is autophagy? When/why does it occur? (2 possibilities)

-The digestion of old or unwanted organelles or other cell structures. -Two types - macrophagy and microphagy. -Macrophagy begins when an organelle or other structure becomes wrapped in a double membrane derived from the ER. The resulting vesicle is called an autophagic vacuole. -Microphagy involves formation of a much smaller autophagic vacuole, surrounded by a single phospholipid bilayer that encloses small bits of cytoplasm rather than whole organelles.

• What,in general, is the SNARE hypothesis?

A proposal for the mechanism by which membranes, particularly vesicular and Golgi or plasma membranes, fuse during, for instance intracellular transport and secretion. The two membranes contain protein complexes, SNAREs, which will become the sites of fusion. Nomenclature derives from an N-ethylmaleimide-sensitive (i.e. sulphydryl-containing) soluble fusion protein (NSF) and soluble NSF attachment proteins (SNAPs); the membrane-associated complexes that recognize SNAPS are soluble NSF attachment protein receptors (SNAREs). The vesicular membrane carries a v-SNARE and the target membrane a t-SNARE. The two SNAREs can form a fusion particle, and with energy input from the hydrolysis of ATP, can cause membrane fusion. Spontaneous fusion is prevented by v- and t-SNARE clamping proteins.

What is a ribozyme?

A ribozyme is a ribonucleic acid (RNA) enzyme that catalyzes a chemical reaction. The ribozyme catalyses specific reactions in a similar way to that of protein enzymes. Also called catalytic RNA, ribozymes are found in the ribosome where they join amino acids together to form protein chains

What is difference between purification and enrichment?

An important thing to note will be that there is cross contamination between pellets. Mitochondria will show up in Pellet 1 as well in Pellet 2. Lysosomes will be in Pellet 2, as well as in Supernatant 2. This shows that the separations made by this technique are not purifications, but relative enrichments of organelles. Enrichment is the goal of differential centrifugation. Enrichment means that the isolated fraction (e.g., pellet 2) has more of the target (mitochondria) than any of the other fractions. This is what we will be measuring in this experiment: the enrichment of mitochondria via centrifugation.

What is the purpose of a KDEL sequence? (see p. 701)

As we saw in chapter 12, a different signaling method is used for proteins whose final destination is the ER. The C-terminus of these proteins usually contains a KDEL sequence, which consists of the amino acids Lys-Asp-Glu-Leu or a closely related sequence. The Golgi complex employs a receptor protein that binds to the KDEL sequence and delivers the targeted protein back to the ER.

What is the purpose of subcellular fractionation?

Because most organelles and macromolecules differ significantly from one another in size and/or density, centrifuging a mixture of cellular components will separate the faster-moving components from the slower-moving ones. This procedure, called subcellular fractionation, enables researchers to isolate and purify specific organelles and macromolecules for further manipulation and study in vitro.

Explain how differential centrifugation is used to isolate various organelles

Centrifugation is a procedure used for the isolation and purification of organelles and macromolecules. This method is based on the fact that when a particle is subjected to centrifugal force by spinning a cellular extract at extremely rapid rates in a laboratory centrifuge, the rate of movement of the particle through a specific solution depends on its size and density, as well as the solution's density and viscosity. The larger or denser a particle is, the higher its sedimentation rate, or rate of movement through the solution. Centrifugation is used routinely in labs throughout the world to separate dissolved molecules from cell debris in suspensions of broken cells, to collect precipitated macromolecules such as DNA and proteins from cell suspensions, and to separate different cellular components based on their sedimentation rate. In essence, a centrifuge consists of a rotor—often housed in a refrigerated chamber—spun extremely rapidly by an electric motor. The rotor holds tubes containing solutions or suspensions of particles for fractionation. Centrifugation at very high speeds— above 20,000 revolutions per minute (rpm)—requires an ultracentrifuge equipped with a vacuum system to reduce fric- tion between the rotor and air. Some ultracentrifuges reach speeds over 100,000 rpm, subjecting samples to forces exceeding 500,000 times the force of gravity (g). Centrifugation is also routinely used for isolating and purifying DNA and proteins. Following cell lysis, insoluble cell debris and organelles are removed by centrifugation, and the soluble cytoplasmic portion containing the DNA or protein is recovered. Treatment with the appropriate precipitating agent makes the DNA or protein insol- uble so that it can be isolated in a second round of centrifugation.

What is the difference between differential centrifugation and density gradient centrigugation?

Density Gradient Centrifugation. Density gradient centrifugation, like differential centrifugation (see Figure 12A-1), is a technique for separating particles such as organelles based on differences in sedimentation rate. For this centrifugation method, however, the sample for fractionation is placed as a thin layer on top of a gradient of solute that increases in density from the top of the tube to the bottom. The effect is illustrated here for three organelles that differ significantly in size and/or density. Subjected to a fixed centrifugal force for five successive time intervals (circled numbers), the organelles migrate through the gradient as distinct bands.

What is the molar concentration of calcium in each of the following locations: ER, cytoplasm of resting cell, and extracellular?

ER stores calcium. ER ~1mM, cytosol ~50nM (very little) in resting cell, outside cell ~1mM. Ratio 10,000:1 btwn ER and cytosol.

Compare and contrast cotranslational import and posttranslational import

Newly-synthesized pro- teins enter the endomembrane system cotranslationally— that is, they are inserted through a pore complex in the ER membrane into the rough ER lumen as the polypeptide is synthesized by the ER-bound ribosome. Most polypeptide synthesis occurs on cytoplasmic ribosomes after mRNAs have been exported through the nuclear pores. Upon arriving in the cytoplasm, these mRNAs become associated with free ribosomes (ribosomes not attached to any membrane). Shortly after translation begins, two main pathways for routing the newly forming polypeptide chains begin to diverge. The first pathway is utilized by ribosomes synthesizing polypeptides destined for the endomembrane system or for export from the cell. Such ribosomes become attached to ER membranes early in the translation process, and the growing polypeptide chains are then transferred across the ER membrane as synthesis proceeds. This transfer of polypeptides into the ER is called cotranslational import because movement of the polypeptide across or into the ER membrane is directly coupled to the translational process. An alternate pathway is employed for polypeptides destined for either the cytosol or for mitochondira, chloroplasts, peroxisomes, and the nuclear interior. Ribosomes synthesizing these types of polypeptides remain free in the cytosol, unattached to any membrane. After translation has been completed, the polypeptides are released from the ribosomes and either remain in the cytosol as their final destination or are taken up by the appropriate organelle. The uptake by organelles of such completed polypeptides requires the presence of special targeting signals and is called posttranslational import.

What is the unfolded protein response

Proteins that repeatedly fail to fold properly can activate several types of quality control mechanisms. One such mechanism, called theunfolded protein response (UPR), uses sensor molecules in the ER membrane to detect misfolded proteins. These sensors activate signaling pathways that shut down the synthesis of most proteins while enhancing the production of those required for protein folding and degradation.

What makes the rough ER rough?

The RER is characterized by ribosomes attached to the cytosolic side of the membrane (the side that faces away from the ER lumen). These ribosomes are what make the RER appear rough.

List the organelles that make up the endomembrane system

The endomembrane system of the eukaryotic cell consists of the endoplasmic reticulum (ER), the Golgi complex, endosomes, and lysosomes (but not peroxisomes). It is associated with both the nuclear envelope and the plasma membrane. The ER lumen is linked to the interiors of the Golgi complex, endosomes, and lysosomes by transport vesicles that shuttle material between organelles, as well as to and from the plasma membrane.

What tole does the smooth ER play in the catabolism of glycogen?

The smooth ER of hepatocytes (liver cells) is also involved in the enzymatic breakdown of stored glycogen, as evidenced by the presence of glucose-6-phosphatase, a membrane-bound enzyme that is unique to the ER. Thus, its presence is used as a marker to identify the ER during subcellular fractionation or to visualize the ER using fluorescent antibodies. Glucose-6-phosphatase hydrolyzes the phosphate group from glucose-6-phosphate to form free glucose and inorganic phosphate (P ᵢ):glucose-6-phosphate + H₂O ⟶ glucose + P ᵢ This enzyme is abundant in the liver because a major role of the liver is to keep the level of glucose in the blood relatively constant. The liver stores glucose as glycogen in granules associated with smooth ER (Figure 12-3a). When glucose is needed by the body, especially between meals and in response to increased muscular activity, liver glycogen is broken down by phosphorolysis (see Figure 9-10), producing glucose-6-phosphate (Figure 12-3b). Because membranes are generally impermeable to phosphorylated sugars, the glucose-6-phosphate must be converted to free glucose by glucose-6-phosphatase in order to leave the cell and enter the bloodstream. Free glucose then leaves the liver cell via a glucose transporter (GLUT2) and moves into the blood for transport to other cells that need energy. Significantly, glucose-6-phosphatase activity is present in liver, kidney, and intestinal cells but not in muscle or brain cells. Muscle and brain cells retain glucose-6-phosphate and use it to meet their own substantial energy needs. The Role of the Smooth ER in the Catabolism of Liver Glycogen. (a) This electron micrograph of a monkey liver cell shows numerous granules of glycogen closely associated with smooth ER (TEM). (b) The breakdown of liver glycogen involves the stepwise removal of glucose units as glucose-1-phosphate, followed by the conversion of glucose-1-phosphate to glucose- 6-phosphate by enzymes in the cytosol. Removal of the phosphate group depends on glucose-6-phosphatase, an enzyme associated with the smooth ER membrane. Free glucose is then transported out of the liver cell into the blood by a glucose transporter in the plasma membrane.

Explain the concept of sedimentation coefficient, and explain the unit "S"

We can express the relative size and/or density of an organelle or macromolecule in terms of its sedimentation coefficient, a measure of how rapidly the particle sediments when subjected to centrifugation. Sedimentation coefficients are expressed in Svedberg units (S), in honor of Theodor Svedberg, the Swedish chemist who developed the ultracentrifuge between 1920 and 1940. The sedimentation coefficients of some organelles, macromolecules, and viruses are shown in Figure 12A-2.

Sedimentation coefficent

a measure of the rate at which a particle or macromolecule moves in a centrifugal force field; expressed in Svedberg units (pg 95, 329)

Membrane assymentry

a membrane property based on differences between the molecular compositions of the two lipid monolayers and the proteins associated with each (pg 167)

Flippases (phospholipid translocator)

a membrane protein that catalyzes the flip-flop of membrane phospholipids from one monolayer to the other (pg 167, 331)

Glycosylation

addition of carbohydrate side chains to specific amino and residues of proteins, usually beginning in the lumen of the endoplasmic reticulum and completed in the Golgi complex (pg 185, 335)

O-linked glycosylation

addition of oligosaccharide units to hydroxyl groups of serine or threonine residues in protein molecules (pg 335)

N-linked glycosylation

addition of oligosaccharide units to the terminal amino group of asparagine residues in protein molecules (pg 335)

Organelles

any membrane-bounded, intracellular structure that is specialized for carrying out a particular function. eukaryotic cells contain several kinds of membrane-enclosed organelles, including the nucleus, mitochondria, Golgi complex, endoplasmic reticulum, lysosomes, peroxisomes, secretory vesicles, and in the case of plants, chloroplasts.

Hydroxylation

chemical reaction in which a hydroxyl group is added to an organic molecule (pg 326)

Homogenization

disruption of cells or tissues using techniques such as grinding, ultrasonic vibration, or osmotic shock (pg 328)

Sarcoplasmic reticulum

endoplasmic reticulum of a muscle cell, specialized for accumulating, storing, and releasing calcium ions (pg 469)

Smooth ER

endoplasmic reticulum that has no attached ribosomes and plays no direct role in protein synthesis; involved in packaging of secretory proteins and synthesis of lipids (pg 89, 325)

Rough ER

endoplasmic reticulum that is studded with ribosomes on its cytosolic side because of its involvement in protein synthesis (pg 89, 325)

Cytochrome P-450

family of heme containing proteins, located mainly in the liver, that catalyze hydroxylation reactions involved in drug detoxification and steroid biosynthesis (pg 326)

Ultracentrifuge

instrument capable of generating centrifugal forces that are large enough to separate subcellular structures and macromolecules on a basis of size, shape, and density (pg 9, 328)

Endomembrane system

interconnected system of cytoplasmic membranes in eukaryotic cells composed of the endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and nuclear envelope.

Centrifuge

machine for rapidly spinning a tube containing a fluid to subject its contents to a centrifugal force (pg 328)

Supernatant

material that remains in solution after particles of a given size and density are removed as a pellet during centrifugation

Pellet

material that sediments to the bottom of a centrifuge tube during centrifugation (pg 329)

Secretory vesicles/granules

membrane-bounded compartment of a eukaryotic cell tat carries secretory proteins from the Golgi complex to the plasma membrane for exocytosis and that may serve as storage compartment for such proteins before they are released: large, dense vesicles. (pg 90, 339)

ER cisternae

membrane-bounded flattened sac of the endoplasmic reticulum

Anterograde transport

movement of material from the ER through the Golgi complex toward the plasma membrane (pg 334)

Retrograde transport

movement of vesicles from Golgi cisternae back toward the endoplasmic reticulum (pg 334)

Endoplasmic reticulum

network of interconnected membranes distributed throughout the cytoplasm and involved in the synthesis, processing, and transport of proteins in eukaryotic cells

Secretory pathways

pathway by which newly synthesized proteins move from the ER through the Golgi complex to secretory vesicles and secretory granules, which the discharge their contents to the exterior of the cell (pg 339)

Glycoproteins

protein with one or more carbohydrate groups linked covalently to amino acid side chains (pg 185, 335)

Sedimentation rate

rate of movement of a molecule or particle through a solution when subjected to a centrifugal force (pg 328)

cis-golgi network

region of the Golgi complex consisting of a network of membrane-bounded tubules that are located closest to the transitional elements (pg 333)

trans-Golgi network

region of the Golgi complex consisting of a network of membrane-bounded tubules that are located on the opposite side of the Golgi complex from the cis-Golgi network (pg 333)

Golgi complex

stacks of flattened, disk-shaped membrane cisternae in eukaryotic cells that are important in the processing and packaging of secretory proteins and in the synthesis of complex polysaccharides (pg 90, 333)

Homegenate

suspension of cell organelles, smaller cellular components, and molecules produced by disrupting cells or tissues using techniques such as grinding, ultrasonic vibration, or osmotic shock

Subcellular fractionation

technique for isolating organelles from cell homogenates using various types of centrifugation (pg 9, 328)

Differential centrifugation

technique for separating organelles or molecules that differ in size and/or density by subjecting cellular fractions to centrifugation at high speeds and separating particles based on their different rates of sedimentation (pg 329)

ER lumen

the internal space enclosed by membranes of the endoplasmic reticulum

Density gradient centrifugation

type of centrifugation in which the sample is applied as a thin layer on top of a gradient of solute, and centrifugation is stopped before the particles reach the bottom of the tube; seperates organelles and molecules based mainly on differences in size (pg 330)

Svedberg units (S)

units for expressing the sedimentation coefficent of biological macromolecules: one Svedberg unit (S) =10^-13 second. In general, the greater the mass of a particle, the greater the sedimentation rate, through the relationship is not linear (pg 329)

Microsomes

vesicle formed by fragments of endoplasmic reticulum when tissue is homogenized (pg 326)

Transport vesicles

vesicle that buds off from a membrane in one region of the cell and fuses with other membranes; includes vesicles that convey lipids and proteins from the ER to the Golgi complex between the Golgi stack cisternae, and from the Golgi complex to various to various destinations in the cell, including secretory vesicles, endosomes, and lysosomes.

Early endosomes

vesicles budding off the trans-Golgi network that are site for the sorting and recycling of extracellular material brought into the cell by endocytosis (pg 339)

Late endosomes

vesicles containing newly synthesized acid hydrolyses plus material fated for digestion; activated either by lowering the pH of the late endosome or transferring its material to an existing lysosome