Biochemistry - Protein Trafficking

Characteristics of chaperone proteins

- ATPases: preferentially bind unfolded proteins in the ADP state - ADP is exchanged with ATP forming an ATP- chaperone complex, which is released from sections of correctly folded proteins - binding/release of chaperone proteins and the ADP-ATP exchange is repeated until the protein assumes its correct conformation

Selectivity of nuclear transport

- molecules <5000 Da, (ions and small proteins) can rapidly diffuse through the pores - larger proteins diffuse at slower rate - Diffusion is inhibited for molecules greater than ~40kDa (require active transport)

Other enzymes that may associate with the nascent proteins during folding in the ER

- protein disulfide isomerase: promotes formation disulfide bonds - peptidyl prolyl isomerase: aids in the folding of proline-containing proteins.

2. Describe the features in the primary structure of proteins that target them for transport to different cellular compartments.

1. Cytoplasmic: no signal 2. Mitochondrial: 30-50 AA pre-sequence w/ amphipathic character (hydrophobic & + charged AA) 3. Nuclear: Lys-Lys-Lys-Arg-Lys patch in the middle; 5 + AA 4. Membrane, secretory: 20-30 AA "signal sequence" w/ core of hydrophobic AA 5. Lysosomal: mannose-6-phosphate

1. mechanisms of mitochondrial protein synthesis

1. Mitochondrial proteins targeted to the mitochondrial matrix; proteins are maintained in an unfolded state in the cytoplasm, assisted by the binding of chaperone proteins Hsp70. 2. targeting sequence interacts with the outer membrane receptor TOM 20/22 and subsequently binds TOM40, the import channel. 3. The outer membrane channel lines up with an inner membrane channel formed by TIM 23/17.

1. mechanisms of protein synthesis carried out by membrane-bound ribosomes>>golgi modifications

1. Proteins for secretion or insertion into the plasma membrane are transported in COPII vesicles from the ER to the Golgi 2. Golgi modifications: trimming of N-linked glycans, addition of O- linked glycans (to serine and threonine residues) phosphorylation of sugar residues and sulfation of both tyrosine residues and some sugar chains.

1. mechanisms of protein synthesis carried out by membrane-bound ribosomes>>glycosylation

1. signal sequence is cleaved & protein is inside the ER 2. proteins are glycosylated with N-linked glycans added to asparagine residues 3. Chaperone proteins aid in protein conformational changes.

Synthesis of mitochondrial proteins once within mitochondrial matrix

1. the targeting sequence is cleaved 2. protein associates with mitochondrial chaperone proteins - bind and release the protein as it assumes its native conformation (along with the hydrolysis of ATP). - proton motive force is required for entry of proteins into the mitochondria - membrane potential across the inner mitochondrial membrane may aid in the insertion of the positively charged residues in the pre-sequence

1. Distinguish the mechanisms of protein synthesis carried out by free and membrane-bound ribosomes.

Free ribosomes - applies to mitochondrial proteins - encoded in the nuclear genome - proteins are translated on cytoplasmic ribosomes and are inserted into the mitochondrial membrane after translation is complete Membrane-bound ribosomes - secretory, membrane, lysosomal proteins - co-translational insertion into membrane

Nuclear import and export

Import into the nucleus: - directed by the presence of sorting signals (nuclear localization sequences -NLS) - NLS recognized by cytosolic proteins ( nuclear import receptors - importins); bind directly or interact through adapter proteins - cargo-bound importins bind the FG Nups and filaments that extend into the cytosol from the NPC - importins repeatedly bind, dissociate and bind to adjacent FG repeats to facilitate movement through the pore. - Once inside the nucleus, the importin releases the cargo and moves back into the cytoplasm Export out of the nucleus: - reverse mechanism - presence of nuclear export signals, similar to NLS - bound by nuclear export receptors (exportins)

1. mechanisms of protein synthesis carried out by membrane-bound ribosomes.

Once protein is inside ER, original signal sequence is cleaved by signal peptidase SRP: Signal Recognition Particle (contains RNA components + proteins) hydrolysis of GTP with SRP release

Mechanism behind insertion of simple membrane proteins

Stop transfer sequence to keep portion of protein within lumen

Nuclear Pore Complexes (NPC)

~30 NPC proteins called nucleoporins (Nups) - 8 Nups form a ring-like structure through which molecules can pass - Additional Nups stabilize the complex or facilitate transport through the pore **Classes** 1. transmembrane Nups - anchor the NPC in the nuclear envelope (also called poms - pore membrane proteins) 2. structural Nups - stabilize the nuclear envelope curvature at nuclear pores and provide scaffolding for assembling other peripheral Nups 3. FG Nups that contribute to the permeability barrier for nonspecific transport and facilitate movement as direct binding sites for transport receptors. So called for their repeats rich in phenylalanine (F) and glycine (G). - structure of the FG Nups have unstructured FG repeats that line the pore & extend in both the cytosolic and nuclear directions - FG repeats act like a fishing net to restrict the diffusion of larger molecules through the pore