3 PATHWAYS OF VESICULAR TRANSPORT

• Secretory pathway: delivers cargo to the plasma membrane.
• Endocytic pathway: uptake cargo from the plasma membrane.
• Retrieval pathway: recycles cellular molecules.

KEY FACTS ABOUT VESICULAR TRANSPORT:

• Compartment lumens mix via the transport intermediate.
• The membrane of each vesicle maintains its orientation.
• If the cell is growing, the secretory pathway is more active than the endocytic pathway.

STEPS IN SECRETORY PATHWAY

• Transport vesicles bud from the ER and carry content away from it to cis side of Golgi.
• Vesicular budding and fusion mediates the transport of cargo through the Golgi stacks, from cis to trans side.
• Cargo exits the Golgi via a transport vesicle on trans side.
• Transport vesicles fuse with plasma membrane or with endosomes (and then lysosomes).

STEPS IN ENDOCYTOTIC PATHWAY

• Early endosome forms from plasma membrane and extracellular materials.
• Early endosome targets cargo to late endosomes.
• Late endosomes then deliver cargo to lysosomes, which degrade cargo.

THE RETRIEVAL PATHWAY TAKES SEVERAL FORMS

• Endosomes can return cargo to the cell surface via recycling endosomes.
• Cargo in early and late endosomes can also return to the Golgi for reuse.
• Vesicles can deliver proteins from the trans face to the cis face of the Golgi.
• Vesicles can return proteins from the golgi to the ER as well.

3 STEPS OF VESICULAR FORMATION

• Cargo selection. Incorporation of cargo into a vesicle is carefully regulated to ensure that only the correct cargo gets transported.
• Vesicular budding. deformation of the hydrophobic membrane bilayer and breaking off of the membrane into a vesicle
• Vesicular targeting and fusion. Highly regulated just like cargo selection.

Cellular compartments are topologically equivalent when:

• Molecules can get from one to another without having to cross a membrane.
• Nuclear envelope, ER, Golgi, transport vesicles, endosomes, lysosomes, and extracellular space = topologically equivalent

KEY PROCESSES IN ER

• Protein glycosylation
• Protein folding

N-GLYCOSYLATION

• Is an ER event
• Is a Co-translational event.
• Occurs on Asn-X-Ser/Thr residues

O-linked glycosylation occurs in the Golgi apparatus

EVENTS OF N-GLYCOSYLATION

• Nascent protein imports co-translationally through translocon.
• Oligosaccharyl transferase transfers oligossacharide from dolichol to N-side group of Asn residue
• 3 sugars trimmed from the 14 sugar oligossacharide as protein exits translocon.
• Chaperones help protein fold in lumen

4 TYPES OF TRANSMEMBRANE PROTEINS

• Type I membrane proteins have a signal sequence at their N-terminus and an internal stop-transfer sequence. They have no cytosolic tail because signal peptidase cleaves it.

• Type II have an internal signal. Their C term is luminal, and their N term is cytosolic.

• Type III have an internal signal sequence. Their C term is cytosolic, and their N term is luminal.

• Type IV are multipass membrane proteins that have multiple internal stop transfer sequences and start transfer sequences.

PROTEINS FOR IMPORT
• Water-soluble proteins
• Transmembrane proteins

KEY MECHANISMS
• Cotranslational: ribosomes continue synthesizing protein as it crosses membrane
• Post-translational: protein imports after it is completely synthesized by cytosolic ribosomes

SIGNAL SEQUENCE
• Hydrophobic sequence (15-60 residues): directs proteins to specific organelles (i.e. ER membrane)

COTRANSLATIONAL IMPORT OF WATER-SOLUBLE PROTEINS

1. SRP (signal recognition particle) recognizes and binds signal sequence on a nascent protein in the cytosol; halts translation
2. SRP (with ribosomal complex) binds SRP receptor
3. SRP released from complex
4. Signal sequence inserts into translocon, translocon opens and translation resumes
5. Signal peptidase cleaves signal sequence and releases protein in ER lumen
6. Chaperones help protein fold correctly

POST-TRANSLATIONAL IMPORT OF WATER-SOLUBLE PROTEINS

1. Chaperones maintain newly synthesized protein’s unfolded conformation in cytosol
2. Translocon/SRP receptor complex recognizes signal sequence
3. Protein enters translocon; chaperone in lumen prevents peptide from sliding back through translocon

CLINICAL CORRELATION

Alzheimer’s and Parkinson’s

• Neurodegenerative diseases that involve improper folding of proteins in endoplasmic reticulum

MITOCHONDRIA

• Generate energy for the cell
• Have numerous metabolic functions
• Comprise mitochondrial proteins (most encoded by nuclear genes)
• Compartmental organization:
  i. Outer membrane ii. Intermembrane space iii. Inner membrane iv. Matrix

IMPORT INTO MITOCHONDRIA
• Prospective matrix protein unfolded in cytosol
• Presequence: signal sequence at N-terminus; positively charged residues
• TOM (translocase of outer membrane): import receptor and translocator
• TIM (translocase of inner membrane): import receptor and translocator
• Protein passes through TOM and enters intermembrane space; then passes through TIM to enter matrix

TOM-TIM alignment: research suggests that TIM tethers to the outer membrane and diffuses laterally until it comes into contact with the TOM/presequence complex

• Protein can enter matrix in energy-dependent manner (presequence cleaved by peptidase and chaperones assist in folding)
• Protein’s transmembrane domain can diffuse laterally out of TIM and embed in inner membrane

CLINICAL CORRELATION
Neurodegenerative disorders
• Often caused by defects in translocases
• Genetic disorder in TIM production results in deafness and dystonia

KEY MECHANISMS OF PROTEIN TRANSPORT

• Gated transport: energy-dependent (nuclear pores)
• Translocation across membranes: mediated by protein translocators (mitochondrion, post-translational and cotranslational import into ER or peroxisomes)
• Vesicular transport: proteins do not cross membranes (from ER to Golgi, through Golgi stacks or to plasma membrane)

PROTEIN TRANSLATION
• Synthesis of almost all proteins begins in the cytoplasm
• 3 possible outcomes depending on signal sequence (or lack thereof):
i. remain in cytosol ii. post-translational import into organelle (mitochondrion, peroxisome, nucleus, ER) iii. cotranslational import to ER

SIGNAL SEQUENCES
• Short amino acid sequences at the terminal end of proteins (address labels)
• 15-60 residues long
• Direct proteins to specific organelles
• Proteins without signal sequences remain in the cytosol

MEMBRANE TOPOLOGY
• Compartments are topologically equivalent if molecules can get from one to another without having to cross a membrane
• ER, Golgi, vesicle, perinuclear space in nuclear envelope are all topologically equivalent
• Mitochondrion, nucleus and peroxisomes are topologically distinct

• Removes misfolded proteins
• Regulates the amount of a protein in the cell at a time

Two major pathways

1. Ubiquitin-dependent pathway
2. Autophagy (targets old and worn-out organelles)

Proteolysis

Break down of proteins into smaller peptides or amino acids

Clinical Correlation

• Pathologic accumulation of misfolded proteins results in various diseases such as sickle cell anemia and certain neurodegenerative illnesses (Huntington’s disease, Alzheimer’s disease, and Creutzfeldt-Jacob disease)

PROTEASOME STRUCTURE – 26S Mammalian proteasome

1. 20S Subunit

• “Central cylinder”
• Comprised of four rings – two inner rings (seven beta subunits each) and two outer rings (seven alpha subunits each)
• Has protease activity

2. 19S Subunits

• “Cap” subunit
• ATPas sites – provide energy for protein degradation and unfolding by using ATP
• Ubiquitin binding sites – recognize proteins tagged for degradation

UBIQUITIN-DEPENDENT PROTEIN DEGRADATION PATHWAY

Protein tagging with ubiquitin

1. Ubiquitin added to cysteine side chain of the E1 protein (ubiquitin-activating enzyme)
2. E1 protein becomes bound to E2 (ubiquitin conjugating enzyme) and E3 protein complex (ubiquitin ligase)
3. Ubiquitin transferred to cysteine side chain on the E2 protein
4. Misfolded protein becomes bound to E3 protein
5. Ubiquitin transferred to lysine side chain on the misfolded protein
6. Repeat steps 1-5 to polyubiquitinate the misfolded protein – polyubiquitin chain is the signal for protein degradation in the proteasome

Proteasomal degradation of the tagged protein

1. Misfolded protein with polyubiquitin chain is recognized by 19S cap of the proteasome
2. Using ATP as an energy source, protein is unfolded, ubiquitin is released, and the protein is translocated into the 20S subunit to be degraded (unknown if unfolding or removal of ubiquitin happens first, but both must occur before translocation can happen)
3. When translocated protein reaches the proteolytic sites, it is cleaved and peptide fragments are released into the cytoplasm for further degradation

PROTEIN SYNTHESIS

Translation

Conversion of mRNA to protein

Codon

Group of three mRNA nucleotides that encode one amino acid

Ribosomal Structure

• 2 subunits each made of protein and RNA:

1. Small ribosomal subunit – has mRNA binding site
2. Large ribosomal subunit

• E-site (Exit site)
• P-site (Peptidyl-tRNA binding site)
• A-site (Aminoacyl-tRNA binding site)

PROTEIN SYNTHESIS STEPS

Initiation

• Initiator t-RNA (methionine) binds to small ribosomal subunit (with help from initiation factors)
• Small ribosomal subunit/initiator t-RNA complex binds mRNA (using 5′ cap to bind the 5′ end)
• Complex scans towards the 3′ end until it locates the start codon (AUG which sets the appropriate reading frame) and the large ribosomal subunit arrives

Elongation

• Current tRNA in the P-site
• New tRNA added to A-site
• Growing peptide chain added to amino acid bound to A-site tRNA
• Large ribosomal subunit shifts 3′ one codon
• Small ribosomal subunit follows – tRNA that was in P-site now in E-site and tRNA that was in A-site now in P-site
• E-site tRNA ejected and new A-site tRNA added

Termination

• When codon in A-site is stop codon (UAG, UAA, or UGA) a release factor is added to the A-site
• Release factor binding releases the peptide chain and causes ribosome to dissociate

POST-TRANSCRIPTIONAL MODIFICATIONS:

• Help with the export of the mRNA from the nucleus
• Protect the mRNA from degradation in the cytoplasm
• Help facilitate proper translation

3 TYPES OF MODIFICATIONS

• 5′ capping
• Poly-A tail
• Intron splicing

5′ capping

• Modified guanine nucleotide
• Added to 5′ end of RNA molecule
• Added during transcription

Poly-A tail

• Multiple adenines added at the end of the polyadenylation signal
• Added to the 3′ end of the RNA

Intron splicing

Intron

Sections of the RNA sequence that are found in between exons

• Introns are removed from the RNA by the spliceosome

Spliceosome

Group of small nuclear RNAs and proteins which join together to recognize and remove introns from transcribed RNA

• Products of intron splice are mature mRNA and circular intron

Alternative splicing allows for one gene to code for multiple, similar proteins

Incorrect splicing is often the cause of the disease Hutchinson-Gilford progeria syndrome

DIFFERENCES BETWEEN PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION

Prokaryotes:

• Only one polymerase isoform
• Occurs in the cytoplasm (since no nucleus)
• RNA polymerase recognizes the promoter sequence
• RNA polymerase recognizes the terminator sequence
• No additional processing of mRNA

CONVERTS INFORMATION IN DNA TO RNA

• Protects original information
• Multiple RNA copies allow for more protein to be produced

DEFINITIONS

Transcription factors

Proteins which bind the promoter and recruit the RNA polymerase

Template strand

Strand of DNA in which the gene to be transcribed is located

THREE STEP PROCESS:

Initiation

• Promoter recognized by transcription factors
• RNA Polymerase II recruited by the transcription factors
• Transcription begins and continues 3′ to 5′ along template strand

Elongation

• Complementary RNA nucleotides added to the growing RNA strand (uracil instead of thymine in the RNA strand)

Termination

• Polyadenylation signal in the transcribed RNA signals proteins to cut it from the polymerase
• Newly formed RNA is called Pre-mRNA because it still needs to be processed
• RNA Polymerase eventually falls off the template strand