Exam 3-Lesson 5 Protein Transport and Folding

• epidermal cell of tobacoo plants

• giant central vacuole that pushes nucleus to edge of cell

Colocalization Experiment with Plasma Membrane marker

• checks whether two things sit in the same place inside a cell.

• label your protein of interest with one fluorescent color and a known reference structure with another, image both, and see how much the two signals overlap.

Signal Peptide

• 15-20 amino acids found at N-Terminus of newly made protein

• shipping label, directs proteins were to go

HDEL Sequence

• four-amino-acid tag, His-Asp-Glu-Leu, at the very C-terminus (the end) of a protein

• ER retention signal (keeps soluble proteins in Endoplasmic Reticulum)

How are molecules transported between cytosol and nucleus?

• nuclear pores!

• small subunits (30-60 kDa) go through pores, ex: RNA & ribosomal subunits go nucleus → cytosol

• Ribosomal subunit proteins/other proteins cytosol → nucleus

• each pore involved in 1,000 translocations per second

Nuclear Basket and Cytosolic fibrils

• proteins

• nuclear basket faces nucloplasm

Nuclear lamina

• signature to animal cells

Why do plants not have nuclear lamina?

• do not have intermediate filaments (which make up nuclear lamina)

• nuclear pore with TEM

Nuclear Importins

• protein used to get proteins into nucleus

• Nuclear localization signal (NLS) recognized by nuclear import receptor (AKA importin)

Nuclear Exportins

• proteins that bring proteins from nucleus to cytosol

• binds nuclear export signals (NES)

What is in the center of the nuclear pore complex?

• proteins binding loosely in gel-like structure (Phe- Gly nucleoporins [FG Nup])

FG Nup

• Fenoalynine Glycine Nucleoporins

• loose gel-like structure of proteins

• important for allowing proteins to pass through nuclear pore

Ras

• used in signaling protein for cell proliferation

• part of MAPKKK pathway

Ran-GTP cycle

• allows proteins to be imported and exported out of nucleus

Ran

• small GTPase, binds GTP or GDP

• Binds GTP when inside nucleus

• Binds GDP while in the cytosol

Ran-GTP Cycle

• Ran GAP (GTPas activating protein) facilitates GTP hydrolysis (in cytosol)

• Ran-GEF (Guanine nucleotide exchange factor) exchanges GDP for GTP in Ran, only in nucleus

How do Ran-GAP and Ran-GEF stay in cytosol and nucleus respectively?

• Nuclear import signal and nuclear export signal (different signal sequences for different proteins)

How does Ran GDP import stuff into the nucleus?

How is Ran GTP involved in export out of the nucleus?

How are proteins transported throughout the cell?

1. Nuclear Pores- proteins transported in folded form

2. Across membranes- proteins transported in unfolded form & refolded after transport (same as bacteria- or accomplished by protein translocators)

3. Vesicles- proteins transported in folded form

Protein translocator

• protein complex that moves proteins into/across membranes

Signal Peptidase

• cleaves off signal peptide in protein

Where is the protein signal sequence located?

N-terminus

(5’ end of mRNA)

What form are proteins usually transported in?

• unfolded

• translocation through both membranes occurs simultaneously if protein is destined for matrix

Where are most proteins for mitochondria located?

• synthesized by ribosomes in cytosol bc most mitochondrial proteins are encoded for in the nucleus

If proteins are transported unfolded, how are they not degraded?

• chaperone proteins bind to aid in transport

How are proteins translocated into chloroplasts?

• extra step b/c of thylakoid membranes

• 1st N-terminal signal peptide cleaved and 2nd signal peptide binds

• directs proteins into thylakoid via 4 different pathways

How are proteins without an ER signal sequence synthesized?

• by cytosolic ribosomes

Signal Recognition Particle (SRP)

• signal (zip code) to guide proteins to correct location

• Think postal worker while signal sequence is mailing address

(technically protein + RNA)- this helps protein be flexible

Polyribosomes

• many ribosomes reading the same mRNA sequence at once

What is cotranslational translocation?

• protein moved across membrane (or inserted in membrane) while still being synthesized

How are soluble proteins translocated into ER lumen?

• soluble= in cytosol

• cotranslational translocation

• signal peptidase cleaves off ER signal sequence (by N-terminus), then degraded while mature protein folds & released into ER lumen

how does SRP use GTP for cotranslational translocation?

which form of energy is used for translation?

GTP

Sec 61

• the core, universally conserved protein-conducting channel (translocon) in the endoplasmic reticulum (ER) membrane of eukaryotic cells that mediates cotranslational translocation.

• Same in bacteria, archaea, eukaryotes

how is post-translational translocation different in bacteria & eukaryotes

Are start and stop transfer sequences hydrophobic or hydrophilic?

• hydrophobic so they stay in membrane

What charge are the residues GENERALLY in the cytosol?

positive

What charge are the residues of the ER lumen GENERALLY?

negative

Microsomes

• mini ERs

• Contains all proteins that would be found in the ER (ex: SRP receptors)

Amino-acyl tRNA

• Charged tRNA (has amino acid bonded to it)

Where must GFP be put on a type 1 protein

• C terminus, otherwise it interferes with protein folding

• ends up in cytosolic end

Type 1 Protein

Type 2 Protein

Type 3 Protein

The ER lumen is topilogically equivalent to…

outside of the cell

Type 4 Protein

• multiple passes of protein

GPI-anchored proteins (Glycosylphosphatidylinositol)

• similar to soluble ER proteins

• start transfer sequence @ n-terminus

• sit on outside of cell surface (anchored to membrane but not actually in-membrane)

• receptors for different molecules

How does adding detergent and protease affect the protein?

• kills/unfolds all the protein

What does the endomembrane system consist of?

What does it mean that vesicles and membranes maintain their orientation?

• non-cytosolic side always faces lumen of compartment or outside of the cell

What is COP1?

Coatomer Protein 1

• vesicular transport between golgi cisternae and from golgi back to ER

What isi COP 2?

ER to golgi

How does clathrin work with endocytosis?

How do SNARES, tethering proteins, and Rab work?

Lysosomes

• only present in animal cells

• Acidic vacuoles in plants and fungi

• Acidic pH, optimal for hydrolases, maintined by active H"+ pumping into lysosomal lumen

• would denature proteins in cell if it broke

If a protein is glycosylated, what can you conclude about its location?

• anywhere in endomembrane system

N-linked glycosylation

• carb chain linked to asparagine residue by ER

• aids in protein folding and function

Are antibodies glycosylated?

Yes! Any eukaryotic organism can be used to make antibodies

What is Endo H?

• Endoglycosidase H

• Recombinant glycosidase (basically specific molecular scissors)

• cleaves off high-mannose oligosaccharides from N-linked glycoproteins

What is Endo D?

• cleaves in ER lumen and golgi