Module 1: Organelles Flashcards
Allosteric Regulation I
Change in protein structure/function due to non-covalent binding by a ligand (eg. calcium, nucleotides, another protein!)
Ca2+ interactions changes calmodulin (CaM) tertiary structure to allow binding to a target protein
Allosteric Regulation II
Guanosine‐triphosphate (GTP) binding changes protein structure to increase enzymatic activity (on)
Guanine nucleotide exchange factor (GEF) – switch out GDP for GTP
GTPase Activating Protein – increase GTPase - which is an enzyme that bind to GTP and hydrolise it to GDP
Post-translational Modifications (PTM)
Covalent modifications that changes protein structure with varied consequences.
* Changed activity
* Target for degradation (protein death)
* Changed cellular location
* Changed structure or organisation
Common protein PTMs:
- Nitrosylation
- Glycosylation
- Methylation
- Acetylation
- Lipidation
- Proteolysis
- Ubiquitination
GLAM-PUN
The Nucleus
DNA within codes most proteins in a cell
Transcription determines the nature of cell/organism
Complex organisation
Nuclear transport essential to link the process of transcription and translation which are separated by nuclear membrane
Nuclear Architecture - Membrane
Membrane—2 membranes and a nuclear lamina (nuclear envelope)
Inner membrane defines nucleus
Outer membrane continuous with rough ER
Lamina is a meshwork of filaments for structure
Nucleolus/nucleoli
Sub-organelle
No membrane
Site of ribosome biogenesis (the process of creating ribosomes in a highly regulated manner)
Formed around regions of DNA encoding ribosomal RNA (rRNA)
Specifically tandem repeated clusters of rRNA genes – Nucleolar Organizer Regions (NOR).
* Hotspot of transcriptional activity (~80% of total RNA in cell is rRNA).
* Thus, nucleoli are genetically defined structures formed as a result of
making ribosomes.
Nuclear Architecture - Nuclear bodies
Membraneless nuclear sub compartments
Concentrated regions of protein and RNA
Associated with transcriptional RNA processing activitie
Chromatin
Packaging of over 2m of DNA within nucleus
Dynamic structure (extended or condensed)
Structure determines gene expression
Regulation of Chromatin Structure
Histone tails (N- or C-term) extending from nucleosome can be targets of several PTMs
HETEROCHROMATIN
Unacetylated: chromatin is highly condensed (transcriptionally inactive)
EUCHROMATIN
Acetylation – chromatin is less condensed (transcriptionally active)
Histone PTMs represents a “histone code” to determine gene expression
(The “histone code” is a hypothesis which states that DNA transcription is largely regulated by post-translational modifications to these histone proteins.)
Proteins that modify histones control chromatin structure and access of DNA to replication, transcriptional and repair machinery
Transcriptional Machinery
- Transcriptional activators bind to DNA to recruit chromatin remodelling complexes to “open up” chromatin structure
- They also recruit a protein bridge (mediator) to help recruit transcription factors to a promoter sequence
- Mediator complex facilitates assembly of the preinitiation complex that includes loading a RNA polymerase (RNA pol II) on DNA
- After initiation, transcription is paused by an elongation factor complex (NELF/DSIF)
- Elongation pause is relieved by phosphorylation and remodelling of the elongation factors by a cdk/cyclin pair (P-TEFb)
The Nuclear Pore Complex (NPC
Spans both nuclear membranes
Sole gateway in/out of nucleus
Allows passive diffusion of small molecules
Human NPCs are large— ~125MDa
Comprised of ~30 nucleoporins (Nups)
Different Nups are repeated 8, 16, 32 or 64 times
Laminopathies - nuclear diseases
Genetic mutations that impact lamins, nuclear membrane proteins connected to lamins or proteins involved in processing or maturation of lamins
- Premature Ageing
- Peripheral and sensory neuropathies
- Familial Partial Lipodystrophy
- Muscular Dystrophies and cardiomyopathies
The ER
Largest continuous membrane structure in a cell
Extensive lace-like network roughly divided into smooth and rough ER
Surface of Rough ER membranes decorated with ribosomes and sites of protein synthesis (translation)
The life of secreted and plasma membrane proteins start at ER: secretory pathway
Nascent proteins folded, modified and assembled within ER lumen
Important roles in protein quality control
ER Organisation
Rough ER has sheet-like structure or “cisternae” (flattened membrane)
Smooth ER has a highly branched, “tubular” morphology
The whole thing (along with nuclear membrane) is one continuous network with common luminal space.
Shaping of ER Membrane
Reticulons are responsible for membrane curvature
Reticulons are inserted into ER membranes in a wedge-like conformation to curve the bilayer
Getting a Protein into the ER Lumen
Requires targeting signal
For secreted proteins, ER signal located at N-terminus of nascent polypeptide
Cleaved following targeting
ER targeting happens at the same time as protein synthesis—”Cotranslational Translocation”
Cotranslational Translocation
Adaptor complex, the signal recognition particle (SRP)
Binds to both the large ribosomal subunit and the signal sequence of the growing peptide
Receptor for SRP in the ER membrane
Translation is halted until the ribosome gets to ER translocon
Docking of the SRP to its receptor opens up a channel allowing the translocation of the newly synthesised peptide
Signal peptidase in the ER cleaves the signal sequence off the polypeptide
Poly peptides fold with the lumen of the ER
ER Membrane Proteins
Classified by topology
Peptide synthesis is unidirectional, so different mechanisms are required for different membrane topologies
Insertion of Type I Membrane Proteins (single transmembrane α-helix protein)
Initial steps are identical to translocation of secreted proteins
Insertion into membrane requires a “stop-transfer anchor” (STA) signal
Hydrophobic amino acids (20-25) that em-beds into lipid bilayer
Insertion of Type II Membrane Proteins (polytopic transmembrane α-helical protein)
DO NOT have a cleavable N-terminus signal sequence
Translation initially occurs in cytoplasm
Internal ER targeting sequences is then recognised by SRP and directed to ER translocon
Internal targeting sequence also doubles as a “Signal-anchor sequence” (SA)
Once SA sequence is embedded, it is moved laterally along the bilayer and ribosome continues cotranslation into ER lumen
Insertion of Type III Membrane Proteins (transmembrane β-sheet protein)
Same topology as Type I but translocation mechanism is similar to Type II
Does not have a cleavable N-term signal sequence
Uses a signal-anchor sequence but positioned very close to N-terminus
Protein Folding and Quality Control
Protein folding and quality control
Newly synthesized proteins in ER undergo several modifications
Fold and assemble properly into mature complex prior to leaving ER (QC) to the Golgi (logistics)
Improperly folded proteins are targeted for destruction
Principal modifications include:
Glycosylation. Covalently linked with oligosaccharides (sugar polymers or glycans)
- Disulphide bond formation
- Protease cleavage (Proteolysis)
- Assembly quaternary structures
Glycosylation
Many secretory proteins and membrane proteins are sugar-modified (glycoproteins)
Transfer of a chain of sugars (glycans) from a precursor catalysed by glycosyltransferases
Glycans can be further modified after initial transfer.
