Exam 4: Lecture 3

Question 1

Polypeptide Folding

Answer 1

• all linear polypeptide chains must be folded into three dimensional structure appropriate for protein in question
• many proteins can spontaneously fold in cell free systems
• rate and efficiency of correct folding reduced when compared to those measured within cell

Question 2

Protein Folding Factors (Chaperone Proteins)

Answer 2

• aid in folding of all proteins within cell
• folding occurs simultaneously with translation
• amino (N) terminal of protein will be folded while carboxyl (C) terminal of protein still being synthesized

Question 3

Rough Endoplasmic Reticulum

Answer 3

• major site for protein folding
• membrane continuous with nuclear envelope
• ribosomes attach to surface via interactions with Ribophorin

Question 4

After Protein Folding

Answer 4

• leave trans face of ER in transport vesicles and shipped to cis face of Golgi
• dock and dump cargo into Golgi
• modified as they travel through Golgi
• modified proteins reach trans face and are put in vesicles and sent to different parts of cell

Question 5

Golgi Apparatus

Answer 5

• several stacks of cisternae
• site for modification and sorting
• some modifications are cleaving proteins or addition of sugar residues

Question 6

Protein Localization

Answer 6

• sorting relies on sequences of aminos acids that act as address label
• compartments include nucleus, ER, Golgi, mitochondria, peroxisomes, and plasma membrane

Question 7

Nucleus

Answer 7

• all replication, splicing, editing and transcription machinery transported here
• NLS consists of 5-10 basic, positively charged amino acids

Question 8

ER

Answer 8

• protein folding factors

- ER retention sequences is only 4 amino acids called KDEL sequence

Question 9

Peroxisome

Answer 9

• breaks down long chains of fatty acids

- proteins residing within all contain three amino acid sequence Ser-Lys-Leu

Question 10

Protein Secretion

Answer 10

• many cells secrete digestive enzymes

- others secrete diffusible ligands to communicate with neighboring cells

Question 11

Secretory Pathways

Answer 11

• costitutive secretion (amylayse)
• regulated secretion (spatzle ligand)
• proteins secreted by these pathways packaged into membrane vesicles that fuse with plasma membrane and dump contents into extracellular space
• constitutive vesicles smaller than those used for regulated secretion
• different vesicles than those used by Golgi

Question 12

Proteins Targeted for Secretion

Answer 12

• by presence of signal peptide at amino terminal of protein
• recognized by Signal Recognition Particle (SRP) located at surface of ER
• signal peptide cleaved from protein while being translated and folded within ER
• untagged protein secreted
• all other proteins in cell have cellular tag so not secreted

Question 13

Localization

Answer 13

-important in determining protein function

Question 14

Method of Determining Localization

Answer 14

• “tag” protein of interest with Green Fluorescent Protein (GFP) and determine where protein resides
• GFP on own resides in cytoplasm but when you fuse it to transcription factor it will be translocated to nucleus
• can then fuse GFP to protein variants and see which chimeric protein translocates to the nucleus and which remains in cytoplasm

Question 15

Why these methods?

Answer 15

• similar types of analysis can be used to determine if a protein is localized to any cellular compartment (i.e. plasma membrane, mitochondria).
• can also determine which domain of protein contains address tag

Question 16

Mitochondria

Answer 16

• energy production center of cell
• proteins destined for it can posses wide range of sequences
• mitochondrial targeting sequences (MTS) contain alternating pattern of hydrophobic amino acids and positively charged amino acids