Cell Biology- Chapter 15

Organelles and Trafficking of Proteins and Vesicles

Intestinal epithelial cells contain all the compartments and organelles of an animal cell

Three modes of transport move proteins around the cell

• Proteins: Nuclear pores

• organelles: Translocators

• everywhere else: vesicles

A protein’s address in the cell is written in the amino acid sequence

Altering a protein’s signal sequence will alter it’s localization

Nuclear pores are the membrane transport complexes of the nuclear envelope

• nuclear pores are multi-protein complexes that span the double membrane of nuclear envelope

Anatomy of a Nuclear pore:

• Cytosolic fibrils are spaced wide enough so not to interfere with proteins entering the nuclear pore, but can be bound by nuclear import proteins during translocation

• Intrinsically disordered fibers project into the pore and form a diffusion barrier that prevents random molecules from entering the nucleus

Nuclear import receptors carry cargo into the nucleus

Proteins bound for the nucleus are recognized by nuclear import receptors in the cytosol.

• The complex of import receptor and its cargo interacts with cytosolic fibrils and then wiggles through the unstructured fibers within the pore

• Once inside the nucleus, the receptor releases its cargo and returns to the cytosol

• There are also nuclear export proteins that carry cargo (like mRNA) out of the nucleus- The mechanism is similar

Ran is a G protein that cycles between the cytosol and the nucleus

• Ran is a monomeric G protein that facilitates recycling of nuclear import receptors

• Recall that G proteins are regulated by whether they are bound to GTP or GDP

• Almost all monomeric G proteins have monosyllabic names that begin with “R” (Ras, Ran, Rac, Rho, Rab, Rap)

• Ran-GAP (GTPase activating protein) in the cytosol promote Ran-GDP formation

• Ran-GEF (Guanine nucleotide Exchange Factor) in the nucleus promotes Ran-GTP formation

Ran facilitates return of nuclear import proteins to the cytosol

• Binding of Ran-GTP to the nuclear import receptor causes it to release its cargo

• The receptor-Ran-GTP complex binds back to the nuclear pore and travels back to the cytosol

• In the cytosol, Ran-GAP converts Ran-GTP to Ran- GDP, which causes it to release the import receptor

• Another transport mechanism brings Ran back into the nucleus

Mitochondrion and chloroplast proteins are unfolded during transport

• Mitochondrion and chloroplast proteins (from nuclear genes) are translated and

produced fully in the cytosol and threaded into their destination through

translocators

Peroxisome proteins are transported directly and shuttled from the ER (by vesicles and transporters)

The endoplasmic reticulum has the most extensive membrane out of any organelle

The same pool of ribosomes translates all nuclear-genome coded proteins

• The status of a ribosome as “free” or “membrane-bound” is fluid and is determined by what specific protein the ribosome is synthesizing

• Because of this, all mRNA transcripts from the nucleus are translated by the same pool of ribosomes

• Mitochondria (and chloroplasts) have their own ribosomes that translate mRNAs transcribed from their own circular genomes

ER signal sequences pause translation until the ribosome docks with the ER membrane

• Proteins that are synthesized at the ER membrane are marked by an ER-localization signal

• This signal is recognized by the signal-recognition particle (SRP), a protein that pauses translation and facilitates docking of the ribosome with a protein translocator on the ER membrane

Soluble ER proteins pass through the translocator completely

• Soluble (as opposed to membrane-bound) proteins formed in the ER have an ER signal sequence at the amino- (N-) terminal.

• The signal sequence (made of hydrophobic amino acids) remains within the ER membrane

• The protein passes continuously through the translocator into the ER lumen as it is synthesized

• At the conclusion of synthesis, a signal peptidase cleaves off the signal sequence and the complete, folded protein is released into the ER lumen.

Single-pass transmembrane proteins partially pass through the translocator

• For some single pass proteins, an N-terminal signal sequence sends them to the ER

  • Translation pushes these proteins through the translocator into the ER lumen, but a second sequence, a “stop-transfer” sequence locks into the translocator and the remainder of the protein is synthesized in the cytosol

  • Cleavage of the signal peptide produces a protein with a luminal face, cytosolic face, and a hydrophobic transmembrane domain (the stop-transfer signal) between them

  • This process produces a single-pass transmembrane protein with its N-terminal

  in the lumen and its carboxy (C-) terminal in the cytosol – Type I Transmembrane

  Protein

  Multi-pass transmembrane proteins have multiple internal transfer sequences

  Multi-pass transmembrane proteins also have a signal sequence, but it’s internal rather than terminal

  • The internal signal sequence leads to insertion of a loop of the nascent protein into the ER translocator

  • The signal sequence acts as the first transmembrane domain of the protein, the transfer-stop sequence act as the second

  • Some multi-pass proteins have many transmembrane domains formed by alternating transfer-start and transfer-stop signals

  • Single pass proteins can contain internal signal sequences, as well.

  • They have no stop signal

  • Such proteins have a luminal C- terminal and a cytosolic N- terminal – Type II Transmembrane Protein

Vesicles begin and end at membranes

• trafficking directs membrane encapsulated molecules around the cell

Clathrin protein forms a basket-like structure to shape vesicles

• triskelion- facilitates cage formation

• clathrin attaches to cargo receptors via adaptins

Secretory and Endocytic Pathways

ER size is dictated by level of secreted/membrane protein production

Many ER synthesized proteins are glycosylated

• the glycocalyx has its origin in the endoplasmic reticulum

• N- glycosylation is the addition of sugar molecules to asparagine residues

• A precursor sugar chain on dolichol is transferred to proteins

• N glycan chains initiated in the ER are trimmed and modified in the Golgi

• Protein substrates for N glycosylation are not permitted to leave the ER until sugars are added

Quality Control mechanisms prevent misfolded proteins from leaving the ER

Accumulation of improperly folded proteins results in ER stress

• efficient folding is assisted by a calcium dependent chaperone called Calnexin

• Inhibition of SERCA depleted ER of calcium and results in misfolded proteins

• Tunicamycin inhibits N-glycosylation and promotes protein retention

• Accumulated proteins cause ER to swell and lead to apoptosis

The golgi is a protein modification and sorting center

• stack of flattened membrane bound spaces

• the cis network faces and receives vesicles from ER

• trans network faces away and sends vesicles to destinations around the cell

• proteins undergo further modifications in the Golgi

Exocytosis can proceed continuously or in response to signals

• regulated and constitutive secretion

Neurotransmitter release by neurons is regulated by calcium

• regulated secretion has been most thoroughly studied in neurons

• Action potential arrival at the axon terminal leads to opening of voltage-gated calcium channels

• influx of calcium promotes vesical fusion with the pre-synaptic membrane

Endocytosis is uptake of extracellular materials

• Phagocytosis requires large-scale membrane and cytoskeletal rearrangements to allow a cell to engulf large materials.

• Not all cells can do this, those that can are phagocytes

• macrophages, neutrophils, B cells, Dendritic sheets are some examples

• phagocytosis can eliminate pathogens

• antigens from phagocytosis are presented on Type II Major Histocompatibility Complexes.

Three Kinds

• receptor mediated

• phagocytosis

• non-receptor mediated

Non receptor mediated is called pinocytosis, exploited by viruses

• an endocytosed virus escapes the clathrin-coated vesicle and begins its life cycle

• capsid proteins interact with plasma membrane proteins

Endocytosis receptor proteins follow one of three fates

• Recycling: The receptor is returned to the plasma membrane to be reused. This is common for receptors involved in nutrient uptake, such as the transferrin receptor.

• Degradation: The receptor is directed to lysosomes where it is broken down. This fate is typical for receptors that need to be downregulated, such as the epidermal growth factor receptor (EGFR) after it has been activated.

• Transcytosis: The receptor is transported across the cell to a different part of the plasma membrane. This process is important in polarized cells, like epithelial cells, where it helps in moving substances from one side of the cell to the other.

Macromolecules directed to lysosomes are digested to their constituent parts

• lysosomes have a complement of digestive enzymes that can break down macromolecules

• lysosomal enzymes work best at acidic pH- the pH is maintained by an ATP-powered proton pump

• simplified metabolites exit lysosome via metabolite transporters.

Exocytosis can proceed continuously or in response to signals

Three pathways transfer materials to lysosomes

• phagocytosis

• endocytosis

• autophagy

Signal pathways can be amplified. Multiple pathways can converge

• Primary transduction is initial activation of intracellular signaling

• most pathways include recruitment of an enzyme during primary transduction

• a single enzyme can produce multiple intracellular messengers that amplify the signal

Signal Pathways can be modulated by positive and negative feedback