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Cell Biology > Week 5 > Flashcards

Week 5 Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

Cytosol

A

The regions inside the plasma membrane but outside all of the membrane bound organelles.

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2
Q

Cytoplasm

A

The area outside of the nucleus and inside the plasma membrane

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3
Q

How much of the total membrane in a eukaryotic cell is found in the plasma membrane?

A

Only about 2-5%.

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4
Q

Signal hypothesis

A

Proteins that leave the cytosol have intrinsic signals that direct them to the appropriate organelle.

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5
Q

Cis-acting signals

A

Signals within the protein that provide its address label.

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6
Q

A sorting signal can be found in which level of structure of a protein?

A

A sorting signal can be part of the primary, secondary, tertiary, or quaternary structure of a protein, or a posttranslational modification.

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7
Q

The protein transport pathway from the cytosol to the nucleus is an example of what type of transport?

A

Gated transport.

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8
Q

Protein transport from the cytosol to the mitochondria, ER, and plastids is an example of what type of transport?

A

Transmembrane transport.

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9
Q

Gated transport

A

Fully folded proteins pass through a large pore (nuclear pore).

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10
Q

Translocation

A

Unfolded proteins are threaded through a small pore (ER translocon). Membrane remains impermeable to small molecules.

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11
Q

Where does translocation occur?

A

In protein transport to the ER, mitochondria, and chloroplasts.

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12
Q

Vesicular transport

A

Proteins move between organelles without crossing a membrane. Carried by small membrane-bound vesicles.

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13
Q

In what type of pathways does vesicular transport take place?

A

Secretory and endocytic pathways.

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14
Q

NLS

A

Nuclear localization signal

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15
Q

How was NLS discovered?

A

Using molecular biology approaches such as necessary and sufficient tests.

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16
Q

How is NLS related to position on the polypeptide?

A

The NLS is position independent and is never cleaved off of the protein after it is used.

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17
Q

Molecular biology approach for transplanting a potential signal sequence onto a reporter protein

A

The DNA sequence coding for the signal is fused in frame with the gene coding for the reporter. The fusion gene is transfected into cells and the fusion protein is expressed.

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18
Q

Sufficiency test for the T-antigen NLS

A

Transplant the putative localization signal onto a cytosolic protein. Ask: Is the signal sufficient to localize the fusion protein to the nucleus?

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19
Q

How is the signal for a sufficiency test for the T-antigen NLS detected?

A

T-antigen NLS is fused to pyruvate kinase (cytosolic enzyme). It can then be detected by indirect immunofluorescence using anti-pyruvate kinase antibodies.

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20
Q

Necessary test for the T-antigen NLS

A

Mutate the putative signal in the context of the full length protein. Ask: Is the signal necessary to localize the protein to the nucleus?

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21
Q

How are the results of the necessary test for the T-antigen NLS evaluated?

A

Each bright structure is a nucleus containing wild-type T-antigen. The mutant T-antigen localizes to the cytosol. The nucleus is the dark region of the cells.

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22
Q

How does T-antigen, or any other nuclear protein, get through the nuclear envelope?

A

The nuclear pore.

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23
Q

Nucleoporins

A

Individual proteins in the nuclear pore

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24
Q

How many unique proteins make up the nuclear pore?

A

~30

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25
Q

Size of the nuclear pore

A

The nuclear pore is a 60 MDa complex

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26
Q

Size of molecules that enter the nucleus by free diffusion

A

< 40 kDa

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27
Q

Size of macromolecules that enter the nucleus by active transport

A

> 40 kDa

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28
Q

Rate of transport into the nucleus through the nuclear pore

A

~ 100 histone proteins/pore/minute

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29
Q

Oily sphaghetti model

A

Model of the nuclear pore that says “greasy” loops of protein extend into the nuclear pore and act as a barrier.

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30
Q

The “postmen” required to deliver cargo into the nucleus

A

Nuclear import receptors and Ran

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31
Q

Ran

A

Small monomeric GTP binding protein

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32
Q

Ran GAP

A

GTPase activating protein

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33
Q

Ran GEF

A

Guanine nucleotide exchange factors.

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34
Q

Role of Ran GEF

A

Kicks out GDP and allows GTP binding

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35
Q

Importins

A

Cargo receptors for nuclear proteins

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36
Q

Role of Ran

A

Ran regulates importin in the process of cargo dissociation.

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37
Q

Localization of Ran-GTP

A

High in the nucleus and low in the cytosol

38
Q

What causes the binding and dissociation of cargo in nuclear transport?

A

Mutually exclusive binding of Ran-GTP and cargo to the import receptor. Therefore, Ran-GTP binding causes dissociation of cargo

39
Q

What causes the binding of cargo in nuclear export?

A

Mutually dependent binding of Ran-GTP and cargo to the export receptor. Therefore, Ran-GTP binding causes association of cargo.

40
Q

What is the role of T-cells, and how are they activated?

A

T-cells help mediate immune responses and are activated by a calcium influx into the cytosol.

41
Q

Effect of calcium influx into the cytosol.

A

Calcium activates a calcium-dependent protein phosphatase called calcineurin, which dephosphorylates NF-AT.

42
Q

NF-AT

A

A transcription factor regulating T-cell response.

43
Q

Effect of immunosuppressive drugs such as cyclosporin A and FK506

A

These drugs inhibit dephosphorylation of NF-AT.

44
Q

hnRNPs

A

Heterogenous ribonucleoprotein particles

45
Q

Role of hnRNPs

A

As RNA is transcribed, it is packaged with hnRNPs. Some of the hnRNP proteins stay in the nucleus while others shuttle back and forth. Nuclear export signal in hnRNP A1 contributes to mRNA export from the nucleus.

46
Q

Nucleolus

A

The region of the nucleus where ribosomes are assembled.

47
Q

What are ribosomes formed from?

A

Ribosomes are formed from a loop of DNA (chromosome) that carries multiple tandem copies of rRNA genes.

48
Q

Composition of the 40S subunit.

A

The 40S subunit has 33 different proteins and one rRNA molecule.

49
Q

Composition of the 60S subunit

A

The 60S has 46 proteins and 3 rRNAs.

50
Q

Where are ribosomal subunits assembled?

A

The subunits are assembled in the nucleus and exported to the cytosol.

51
Q

What type of reactions occur in the peroxisomes of mammals?

A
  • Oxidative and peroxidative reactions (detox).
  • RH2 + O2 –> R + H2O2
  • Catalase converts 2 H2O2 –> 2 H2O and O2
  • B-oxidation of lipids, particularly very long chain fatty acids (breakdown to 2-carbon units)
52
Q

Role of peroxisomes for plants

A
  • Photorespiration in leaves
  • Oxidation of lipids (breakdown of acyl chains 2 carbons at a time) for biosynthetic use
  • Glyoxylic acid cycle in plants allows conversion of fats to sugars.
53
Q

Before GFP was discovered, what was firefly luciferase used for?

A

It was developed for use as a reporter in gene fusion experiments.

54
Q

General reaction for using luciferase to make light

A

Luciferase + luciferin + ATP -> light

55
Q

How was luciferase used to discover a peroxisomal targeting signal?

A
  • Firefly luciferase localized to peroxisomes when expressed in mammalian cells, plants, or yeast (co-localizes with catalase)
  • Necessary and sufficient tests determined that the C-terminal SKL is luciferase’s peroxisomal targeting signal (PTS)
56
Q

Pichia pastoris

A

Methanol-eating yeast

57
Q

Results when Pichia are grown on methanol as the sole carbon source

A

They are filled with peroxisomes and express high levels of methanol oxidase, a peroxisomal protein.

58
Q

What is required for Pichia to grow on either methanol or fatty acids (oleic acid) as the sole carbon source?

A

Peroxisomes

59
Q

What technique was performed to identify mutant yeast that could not grow on either methanol or oleic acid as the sole carbon source?

A

Genetic screens

60
Q

Result of the genetic screens on mutant yeast

A

These screens identified the pex (peroxin) mutants, many of which exhibited peroxisome “ghosts” by electron microscopy.

61
Q

How were the PEX genes identified in yeast analyzed?

A

They were cloned by complementation and the proteins were characterized.

62
Q

What was the rationale/hypothesis for the Pichia pastoris experiments?

A

Because the biochemical pathways for methanol and fatty acid breakdown are very different, the assumption was that a single gene mutation that disrupted both of these pathways would likely disrupt peroxisome biogenesis (the one thing both pathways have in common is that they occur within peroxisomes.)

63
Q

What were the four mediums that Pichia were grown on?

A
  1. Glucose
  2. Methanol
  3. Oleic acid
  4. Ethanol
64
Q

PTS1

A

The C-terminal SKL sequence

65
Q

PTS1R

A

The receptor that binds the SKL sequence (AKA Pex5)

66
Q

Describe the model for peroxisomal protein import.

A

The receptor (PTS1R or Pex5p) brings cargo to the peroxisomal membrane where other Pex proteins form a translocation pore and mediate the release and recycling of the receptor. Fully folded catalase can cross the peroxisome membrane.

67
Q

Zellweger Syndrome

A

A type of peroxisome biogenesis disorder (PBD). Physical presentation: High forehead, epicanthal folds, and hypoplasia of supraorbital ridges and midface.

68
Q

Involvement of fibroblasts in PBDs

A

Fibroblasts from many patients are deficient for uptake of peroxisomal proteins.

69
Q

Describe the mitochondria membranes.

A

Mitochondria have an inner and outer membrane that defines two distinct compartments, the intermembrane space and the matrix

70
Q

Role of mitochondria and how they accomplish it

A

ATP generation. Electron transport in mitochondria and photosynthesis in chloroplasts. Also involved in lipid synthesis

71
Q

Describe the chloroplast membranes.

A

Chloroplasts have three membranes: outer, inner, and thylakoid that define the intermembrane space, the stroma, and thylakoid lumen.

72
Q

What is the mitochondrial protein signal sequence?

A

An N-terminal alpha-helix with basic residues on one face. The secondary structure is important for this signal.

73
Q

Role of TIMs and TOMS

A

TIMs and TOMs form the translocation channel and signal sequence receptors for mitochondrial protein import.

74
Q

TOM translocation

A

Across the outer membrane

75
Q

TIM translocation

A

Across the inner membrane

76
Q

Upon arrival in the mitochondrial matrix, what often happens to the signal sequence?

A

The sequence is cleaved by signal peptidase.

77
Q

What are the energy requirements for mitochondrial protein import?

A

Hsp70: Heat shock protein of 70 kDa. ATPase needed to maintain the unfolded state of the precursor in the cytosol. A mitochondrial Hsp70 is also required in the matrix to facilitate unidirectional translocation. The electrochemical proton gradient, generated by the electron transport chain, is also required to drive the matrix protein across the inner membrane.

78
Q

What encodes mitochondria and chloroplast proteins?

A

Mitochondria and chloroplasts have their one genome, but the majority of their proteins are encoded by nuclear genes.

79
Q

Where are nearly all proteins that are secreted synthesized?

A

Nearly all proteins that are secreted, or are part of the nuclear envelope, ER, Golgi, secretory vesicles, plasma membrane, endosomes, and lysosomes are initially synthesized at the ER.

80
Q

Where is most of the phospholipid and sterol for the entire cell synthesized?

A

The ER. The ER is a biogenic membrane.

81
Q

What is the lumen of the nuclear envelope, ER, Golgi, secretory vesicles, endosomes, and lysosomes topologically equivalent to?

A

The outside of the cell.

82
Q

Which organelles compose the secretory pathway?

A

The ER, Golgi, and secretory vesicles.

83
Q

Which organelles compose the endocytic pathway?

A

The endocytic vesicles, endosomes, and lysosomes.

84
Q

What pulls the ER out of the nuclear envelope?

A

The ER is pulled out of the nuclear envelope along microtubule tracks by a plus end directed motor protein (a kinesin).

85
Q

MTOC

A

Microtubule organizing center

86
Q

What concentrates the Golgi at the MTOC?

A

The Golgi is concentrated at the MTOC by a minus end motor.

87
Q

When calcium is high in the cell, what does it bind to? What effect does this have?

A

Calcineurin. This initiates the regulation of nuclear transport.

88
Q

Nucleolus

A

A protein-rich region where ribosomes are produced. Stains darkly on TEM micrograph.

89
Q

What happens to the ER in a cell lysate?

A

The ER fragments into microsomes in a cell lysate.

90
Q

Role of smooth ER

A

Produces most of the lipids (sterols and phospholipids) for the cell. Detox of hydrophobic compounds.

91
Q

What determines the density of an organelle?

A

The protein to lipid ratio. The higher the protein content, the higher the density.

92
Q
A

Cell Biology (17 decks)

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