Exam 3: Ch 9 Isolating Organelles & Perturbing Cell Functions

Question 1

receptor-mediated endocytosis

Answer 1

take in larger molecules than ions by forming coated pits that pinch off to form endosomes

receptors bring in specific molecules

Question 2

lysosome

Answer 2

organelle with an acidic degradative environment

full of enzymes called acid hydrolases in the lumen

Question 3

endoplasmic reticulum

Answer 3

largest internal membrane system

flattened membrane bound sacs and tubules

Question 4

smooth ER

Answer 4

site of fatty acid and phospholipid synthesis

Question 5

rough ER

Answer 5

ribosome studded ER

site of membrane protein and secreted protein synthesis

Question 6

golgi apparatus

Answer 6

stack of flattened membranes called cisternae

proteins are modified and sorted before transport to final destination in plasma membrane or endosomes

Question 7

secretory pathway

Answer 7

protein synthesis in ER –> golgi –> secretion

includes memb. bound proteins

Question 8

peroxisome

Answer 8

spherical organelle with oxidase enzymes

use molecular oxygen to oxidize toxins

Question 9

initial step in purifying organelles

Answer 9

release cell’s contents by rupturing plasma membrane/cell wall

blender (sonication), osmosis, pressurized tissue homogenizer

Question 10

_______ can separate many types of organelles

Answer 10

differential centrifugation

makes lots of pelleted fractions

Question 11

equilibrium density gradient centrifugation

Answer 11

separate cellular components by density

Question 12

organelle-specific _____ are useful in preparing highly purified organelles

Answer 12

antibodies

use of monoclonal Ab to specific organelle proteins

ex. clathrin

Question 13

clathrin monoclonal ab

Answer 13

selectively bind clathrin bound vesicles in a crude prep of membranes

isolated via low speed centrifugation

Question 14

fat and muscle cells contain a glucose transporter ______ that is localized to the memb of vesicles

Answer 14

GLUT4

add insulin and vesicles fuse with plasma memb. and increase # of GLUT4

purify with monoclonal Ab to GLUT4 protein

Question 15

if no Ab specific for the organelle under study is available, use

Answer 15

epitope tag then monoclonal Ab

Question 16

3 steps to identifying all the proteins in an organelle

Answer 16

obtain pure organelle

identify all the sequences of the proteins in the organelle

have a genome sequence to identify the proteins from which all the peptides came

Question 17

discovery of a new drug?

Answer 17

chemical library used to search for chemicals that inhibit a specific process

uses microscopy

ex. monastrol inhibits spindle assembly (used as anti-tumor agent)

Question 18

RNAi method

Answer 18

suppress expression of gene with miRNA (block translation) or siRNA (degradation)

function in nucleus with target mRNA (imperfect (miRNA) or perfect (siRNA))

RISC: RNA induced silencing complex

Question 19

siRNA knockdown (customized siRNA-RNAi)

Answer 19

identify sequence in target mRNA and synthesize siRNA to add

if perfect binding, no target protein made

Question 20

shRNA

Answer 20

short hairpin RNA cleaved by dicer to form siRNA

alternative knockdown strategy by adding DNA constructs that generate siRNA when transcribed

once shRNA is expressed, siRNA always made

Question 21

genomic screen with siRNA in fruit flys

Answer 21

explore effects of using siRNA to suppress each protein-coding gene in fruit flys