Chapter 8 Flashcards
What does the endomembrane system include?
Organelles such as ER, Golgi, endosomes, lysosomes, and vacuoles
How does one study the endomembrane system?
Using autoradiography and electron microscopy (kills cells)
- Green fluorescent protein labelling (living cells)
How does one use autoradiography to study the endomembrane system? What discovery was made through this method?
Incubate tissue in solution containing labeled amino acids. These amino acids are then taken up by cells and incorporated into proteins. When tissues are viewed, you see large black dots where proteins are localized. This is how it was discovered that proteins are synthesized in the ER
_____ of the endomembrane system are part of an integrated network in which materials are shuttled back and forth
organelles
How are materials shuttled between organelles?
In membrane bound transport vesicles
Name and describe the pathways through the cytoplasm
Biosynthetic pathway - Synthesis, modification and transport of proteins
Secretory pathway - When proteins are discharged (secreted) from the cell either in continuous fashion (constitutive secretion) or in response to a stimulus (regulated secretion)
Endocytic pathway - Move from outer surface of cell to compartments such as endosomes
_____ is a method used to visualize biochemical processes using radiolabeled materials exposed to photographic film
Autoradiography
Describe how green fluorescent protein is used to study cytomembranes
IT is a protein isolated from jellyfish which emits green fluorescent light. GFP needs to be added to the protein of interest. A GFP-DNA chimera enables observe protein synthesis.
_____ determine where proteins are targeted/where they will go
Sorting signals
If GFP shows that the protein is in the golgi, how does one prove that it IS, in fact, the golgi?
You can prove it by doing a second run with GFP tagged “marker proteins” (proteins known to be present in the Golgi. You can also do “z” section - series of sections with x,y,z axes
Does centrifugation break open cells?
NO!!
How are organelles separated from one another for study?
Homogenizing cells, then performing subcellular fractionation. A series of centrifuge cycles can separate out “microsomes”, which are endomembrane vesicles
How can one separate rough and smooth ER?
homogenize solution until microsomes have been separated out. Then undergo sucrose gradient centrifugation. smooth have a low density, rough have a high density.
What is sucrose gradient centrifugation?
Gradient of increasing sucrose concentration established in centrifuge tube with ER pellet on top. After centrifugation, smooth ER should be on top and rough on bottom
________ systems do not contain whole cells and have provided information about the roles of proteins involved in membrane trafficking
Cell-free systems
Why is cell-free system research so helpful?
One can develop mutants for endomembranes, observe cell-free synthesis of proteins
Why is the study of mutant cytomembranes so important?
They provide insights about the functions of normal gene products.
Isolation of proteins from _____ has led to identification of homologous proteins in mammals
yeast
Describe the findings from studying yeast mutant ER
Mutation in gene for vesicle formation (Sec12) prevented vesicles from forming, building up ER volume.
Mutation in gene for vesicle fusion (Sec17) prevents vesicle fusion, resulting in a bunch of unfused vesicles
______ is the process in which cells produce small RNAs that bind to specific mRNAs and inhibit the translation of these proteins
RNA interference
How do scientists figure out which genes are involved in transportation of proteins?
By identifying small interfering RNAs that interfere with those process
Describe the components of the endoplasmic reticulum
Rough ER (covered in ribosomes), smooth ER (devoid of ribosomes). The luminal/cisternal space is inside the ER membrane, and the cytosolic space is outside.
Describe the rough ER
Composed of a network of flattened sacs called cisternae. Continuous with outer membrane of nuclear envelope and has ribosomes on cytosolic surface. Mostly functions in protein synthesis. Polarity in RER sometimes reflects flow from synthesis to discharge of proteins
Describe the smooth ER
Not covered in ribosomes. Functions include synthesis of steroid hormones (in endocrine cells), detoxification in liver, sequestration of calcium ions in muscle cells