yeast models in membrane trafficking Flashcards
why is membrane trafficking necessary?
- Compartmentalisation allows more complexity for a cell
- Enzymes can modify specific subsets of proteins in certain environments e.g through glycosylation and proteolytic cleavage
- For sequential modifications, proteins need to be exposed to distinct sets of enzymes
- retrieval of proteins back to their ‘resident’ compartment
Secretory/Exocytic pathway:
(biosynthetic)
proteins are modified as they travel through
ER → Golgi → PM/endosome/lysosome
function of transgolgi network
sorting network to be secreted in right place
Endocytic pathway:
(recycling or degradative)
cell surface → endosome → Golgi/ER/lysosome
how are proteins modified in er/golgi
oligosaccharide addition (many sugars)
Both N-(asparagine) and O-(OH) linked glycosylation can occur on proteins
asparagines = sites of modification
effect of oligosaccharide addition on proteins
sugars have big impact on overall protein structure due to weight - changes conformation
function of glycosylation
- assist in folding
- as a ligand = intracellular for trafficking and sorting
= Outside the cell for interactions with extracellular matrix and with proteins/sugars on other cells
what makes organisms useful to identification of faulty genes in trafficking
- Simplicity - trafficking occurs on a cellular scale so a single celled organism is likely to provide information.
- Analysis of specific types of secretion e.g regulated secretion, would need a model system that is able to perform this function.
advantages of using yeast
- amenable for genetic studies (can grow as haploid and diploid cells)
- entire genome sequence known since 1996
- limited gene diversity (both ±)
- fundamental pathways conserved
disadvantages of using yeast
- limited cell-cell contact so unlikely to be informative about multicellularity
- small (5µm), so high resolution imaging studies of intracellular compartments is difficult.
- Has a cell wall which can preclude some types of studies
- e.g microinjection
role of ERGIC
- material is trafficked here after ER
- the compartment then changes rather than individually trafficked then things are const. secreted
key experiment in identifying secretory pathway in yeast
novick and schekman
- stages to investigate
- movement in and out
- things docking with plasma membrane
- no transport = cell density increases
how many genes were identified as important in trafficking
- 23 were identified by grouping mutants with similar phenotypes
- so at least 23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membrane.
function of class a sec genes
class a = transport to er
function of class b sec genes
budding of vesicles to go to golgi
function of class c sec genes
fusion of transport vesicles with golgi
function of class d sec genes
transport from golgi to secretory vesicles
function of class e sec genes
transport from secretory vesicles to cell surface (docking)
where is direction of trafficking decided
trans golgi network
what is endocytosis
Endocytosis is the process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endo-lysosomal membrane system.
importance of endocytosis
- Retrieval of molecules that formed part of the secretory vesicle for recycling - labelling to bring back to specific regions
- Downregulation of signals
- Remodelling cell surface lipid and proteincomposition depending on environment
- endocytosis is also a means of entry into cells for many pathogens and toxins
stages of endocytic pathway
- Plasma membrane envaginates to form endocytic vesicle
- Endocytic vesicle to early endosome
- Early endosome to late endosome (MVB) or recycling to the plasma membrane
- Late endosome to Golgi or vacuole
major function of the lysosome (vacuole)
degradation of extracellular material taken up by endocytosis as well as certain intracellular components by a process termed autophagy.
how does lysosomal protein sorting occur
The lysosome’s resident enzymes are transported to the lysosome through the secretory pathway. At the late Golgi compartment (Trans Golgi Network), they are sorted into a pathway destined for lysosomes rather than the plasma membrane.
what is CPY and why is it used
- Carboxypeptidase Y (CPY) is a vacuolar enzyme normally transported to the lysosome having been trafficked through the ER and Golgi.
- As with a-factor CPY is glycosylated and proteolytically cleaved at different stages. This helps us follow its progress
Vacuolar mutants are divided into classes depending on what
depending on the stage at which they appear to block the route to the vacuole
what is a multivesicular body
organelle packed full of material and deposits this into a lysosome
destinations of transport from golgi network
- To plasma membrane
- To early endosome
- To late endosome/MVB
- To vacuole
CPY signalling mechanism
- in late golgi gets recognised by receptor protein (vps10)
- vps10 is sorted into vesicle to traffic to late endosome
- when bound is recognised by gga1 and gga2
- cpy dissociates from vps10 at late endosome and transports to vacuoles where it is cleaved to generate mature form
- Vps10 is retrieved to the late Golgi through a specific aromatic -based signal in its protein sequence (YSSL, FYVF).
- vps10 recognised by specific tail