lecture 1 - eukaryotic diversity and cell structure Flashcards
How do transcription and translation differ between prokaryotic and eukaryotic cells?
In prokaryotic cells transcription of DNA into RNA and translation of RNA into protein are coupled and occur at the same time and in the same place.
In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm - allows multiple layers of regulation e.g during development, in response to environmental cues – but necessitates shuttling of components across nuclear envelope).
nuclear envelope has selective openings for traffic in/out. There are many examples of traffic across the nuclear envelope e.g. ribosome assembly.
Storage of genomic material in a nucleus is a major difference between eukaryotic and prokaryotic cells.
Describe the structure and function of ribosomes
Ribonucleoprotein complexes
Large MWt ~5 x 106
Translate mRNA -> protein
Assembly:
Translation of mRNA: cytoplasm
Assembly of polypeptides onto nascent rRNA: nucleolus
Subunits transported to cytoplasm for function
Ribosomes are the molecular machines responsible for reading genetic information contained within mRNA and building proteins (translation which occurs in the cytoplasm).
Ribosomes are large ribonucleoprotein complexes– containing numerous proteins (~70 in humans) assembled on RNA scaffolds - organised into two subunits – large and small.
Ribosomal subunits are assembled in an area of the nucleus known as the nucleolus before being transported across the nuclear envelope to function in the cytosol.
How are ribosomes assembled?
Transcription of rRNA and mRNA: nucleus
Translation of mRNA: cytoplasm
Assembly of polypeptides onto nascent rRNA: nucleolus
Subunits transported to cytoplasm for function
Synthesis and assembly of ribosomes requires transport of multiple components across the nuclear envelope in both directions.
Transcription of genomic DNA takes place in the nucleus; this is the case for both ribosomal RNA and messenger RNA (that encodes the ribosomal proteins) – but whereas the ribosomal RNA stays in the nucleus for ribosome assembly, mRNA is transported out of the nucleus into the cytoplasm where translation occurs – thus ribosomal proteins are made in the cytoplasm before being transported into the nucleus for assembly onto the ribosomal RNA scaffold. Once assembled, large and small ribosomal subunits are transported out into the cytoplasm where they function to translate mRNA (which has also been transported out of the nucleus to the cytoplasm following transcription).
How do ribosomes function?
Free or membrane bound (association depends on protein being translated)
Once ribosomal subunits have been assembled and are in the cytoplasm– they function to translate messenger RNA into proteins.
All ribosomes are assembled from a pool of ribosomal subunits in the cytoplasm with one large subunit and one small subunit coming together to translate mRNA.
As translation proceeds, the nascent polypeptide grows and emerges from the ribosome tunnel.
If the protein being translated is to stay in the cytosol, the ribosomes keeps moving along the message and as it does so the polypeptide chain grows and more ribosomes attach to create a polysome. Once a stop codon is reached the polypeptide is released and the ribosomal subunits recycle back in to the common pool.
If the message encodes an integral membrane protein, or a protein to be secreted from the cell (e.g. hormones, neurotransmitters), the first bit of sequence to emerge from the ribosome (the N-terminus of the protein) forms what is known as a signal peptide that gets recognised by a factor called the signal recognition particle (SRP). SRP halts translation and directs the ribosome, along with the mRNA and the emerging polypeptide to an organelle called the endoplasmic reticulum (ER). Once the ribsosome has attached to the ER membrane (via the SRP-receptor) translation starts again and the growing polypeptide crosses the membrane through a specialised channel - and in the case of a protein to be secreted it gets released into the lumen of the ER, or integral membrane proteins become incorporated into the ER membrane.
Describe how recognition of signal peptide facilitates entry of proteins into the secretory pathway
If the N-terminus of a protein is a signal peptide (typically a stretch of hydrophobic amino acid residues), the ribosome translating the protein is directed to the endoplasmic reticulum -the point of entry of the secretory pathway.
Prior to secretion from the cell proteins move through the Golgi apparatus.
Secretion can be constitutive (i.e. happens all the time) or regulated (controlled in response to environmental factors e.g. secretion of hormones, release of neurotransmitters).
A major function of intracellular compartmentalisation is that it allows the cell to coordinate complex biochemical reactions such as those required for modifying proteins after they have been translated.
Describe glycosylation
One of the many post-translational modifications that proteins receive as they travel through the secretory pathway is the addition of carbohydrate in the process of glycosylation.
Glycosylation is a very diverse modification that serves many functional and structural purposes. Its diversity arises from the way it is built up using numerous different combinations of a relatively small number of sugar molecules assembled into branching networks on proteins – the synthesis of these chains is very complex, and highly regulated requiring a large number of enzymes to form the different sugar linkages.
Different glycosylation enzymes catalyse addition of sugar moieties to proteins during complex biochemical reactions requiring coordination to produce the large diversity of glycoproteins present in eukaryotic cells
These different glycosylation enzymes are housed in different membrane bound compartments
Proteins encounter these in a defined order as if progressing along a conveyor belt
These reactions have to be carried out in a specified order. To achieve this the cell contains subsets of the enzymes in separate membrane-bound organelles that proteins travel through as they journey through the secretory pathway.
The first of these is the rough ER where proteins receive what is called core glycosylation (covalent attachment of the first sugar molecular onto the polypeptide). Once the protein is ready to leave the ER it moves on through the other compartments that make up the Golgi apparatus receiving sequential modifications as it goes along.
Describe the structure and function of the Golgi apparatus
Flattened stack of individual cisternae, each with unique complement of macromolecules
Each cisternae of the Golgi apparatus is a separate membrane-bound orgnaelle with its own set of macromolecules, including a distinct set of glycosylation enzymes as depicted on the last slide – and proteins travel through these cisternae entering at the cis side from the ER, on through the medial cisternae receiving sequential modifications as they go through.
Last cisternae of the Golgi is continuous with the trans Golgi network or TGN
What is the trans Golgi network?
The trans Golgi network (TGN) forms part of the biosynthetic (secretory) and endoyctic pathways, and serves as a sorting station as part of the endosomal system
The trans Golgi network (TGN) is a highly dynamic series of interconnected tubules and vesicles
It is in the TGN that proteins which have trafficked through the ER and the Golgi are packaged into secretory vesicles that fuse with the plasma membrane to secrete cargo out of the cell.
The TGN is also part of the endocytic pathway.
Endocytosis is:
where a portion of the plasma membrane invaginates and pinches off to form a membrane-bounded vesicle called an endosome
used by cells to ingest substances from outside the cell and to remove molecules from the plasma membrane.
Endocytosed material can be delivered to a number of different compartments within the cell including lysosomes and the TGN (note how the TGN receives material from different pathways, including biosynthetic and endocytic and serves to sort them to their required destination, be that out of the cell, or to other intracellular compartments including the lysosome)
Lysosomes contain degradative enzymes and provide a good example of why eukaryotic cells are compartmentalised. It is important that these degradative enzymes are sequestered away from the rest of the cell so that they don’t degrade cellular components other than those delivered to the lysosome (when they are no longer required by the cell). In addition these enzymes have evolved to function only at low pH (the inside of the lysosome is acidic) and therefore if any of the degradative enzymes end up in the cytosol by mistake they won’t cause problems as they won’t function properly under the neutral pH conditions of the cytosol.
What are the advantages of compartmentalisation of eukaryotic cells into discrete membrane bound organelles?
Coordination/regulation of complex biochemical reactions (e.g. secretory pathway) such as glycosylation
Sequestration of components
e.g. lysosomes/peroxisomes - a major function of which is to break down long chain fatty acids (they contain enzymes to do this– during this process hydrogen peroxide is produced – and used subsequently to oxidise and thus detoxify various substrates including alcohol – hydrogen peroxide is very reactive and it’s therefore important to keep it sequestered away from the rest of the cell.
Energy generation - (mitochondria/chloroplasts)
mitochondria and chloroplasts generate electrochemical gradients across their membranes during respiration and photosynthesis. These energy generating reactions are only possible because the membrane of these organelles are tightly sealed.
Describe how molecules are moved between organelles by vesicular transport
Although compartmentalisation gives eukaryotic cells numerous advantages it also presents the cell with a major challenge – i.e. although different organelles are self contained there has to be a high level of communication between them. Communication occurs via exchange of material.
Consider the secretory pathway – proteins move from the ER, through the Golgi apparatus before being secreted out of the cell.
This occurs by means of vesicular transport whereby a portion of one organelle (donor) pinches off to form a transport vesicle that fuses with another (the acceptor or target) organelle delivering its contents (both membrane and lumenal).
How do we know the order of the secretory pathway? How do we know that proteins travel from the ER and then through the Golgi before being secreted?
Electron microscopy techniques in the early 20th century gave a good descriptive picture of different organelles inside the cell and fractionation experiments (separating cells into membrane and non-membrane constituents) had revealed that before they’re secreted from cells proteins are associated with membranes
What is autoradiography?
bio-analytical technique that can be used to visualise the distribution of molecules labelled with radioisotope within a biological sample.
can localise molecules within tissues, cells, organelles, and supramolecular complexes.
sensitive and quantitative.
works by incorporating radioisotopes into molecules – the energy emitted by these can then be detected.
What is pulse chase labelling?
incorporate radioactive isotopes into molecules syntheszied during a defined time period.
Radio-labelled compound added to system of choice for a brief period of time (the ‘pulse’) after which the ‘chase’ is initiated by either washing the sample with buffer to remove the isotope and/or adding excess non-labelled compound.
used to follow the dynamics of cellular processes and pathways; e.g. changes in a protein’s localization and/or levels over time.
What were Palade’s experiments?
Some cells secrete a lot more proteins than others – for example liver cells and pancreatic cells are considered professional secretory cells – and are deemed good models to study the secretory pathway.
Palade used slices of pancreas for his experiments.
Slices of pancreas in culture synthesize and secrete enzyme.
Add 3H-leucine – becomes incorporated into newly synthesized proteins.
After 10’– wash the slices, and initiate ‘chase’ (excess unlabeled leu)
Perform autoradiography after 0, 10 and 60 minutes
Following pulse-chase labeling, proteins were first detected at the rough endoplasmic reticulum, then the Golgi apparatus, then none was detected intracellularly (but appeared in the medium) indicating that before being secreted from the cell the proteins travel through the ER and then the Golgi.
Having established the order of the secretory pathway Palade wanted to know how transport between these compartments occurred. He noticed occasional ‘spots’ (of radiolablled protein) in small vesicles between different membrane-bound compartments and formulated the hypothesis of vesicular transport.
