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Polypeptide Folding

-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


Protein Folding Factors (Chaperone Proteins)

-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


Rough Endoplasmic Reticulum

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


After Protein Folding

-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


Golgi Apparatus

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


Protein Localization

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



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



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



-breaks down long chains of fatty acids
-proteins residing within all contain three amino acid sequence Ser-Lys-Leu


Protein Secretion

-many cells secrete digestive enzymes
-others secrete diffusible ligands to communicate with neighboring cells


Secretory Pathways

-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


Proteins Targeted for Secretion

-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



-important in determining protein function


Method of Determining Localization

-"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


Why these methods?

-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



-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