09-11-07: First draft, first section

My supervisor gave his opinion on the first section of my literature review draft. There are many English writing faults. The most interesting one is that I am germanish - I put loads of adj. in front of a noun.
Anyways, this beginning is not bad. I am making conjugates using N-succinimidyl 3-(2-pyridyldithio) propionate. This technique is quite old (first described in 1978) but should work fine. EM sectioning is not so bloody to me now, my hands are not bad, good buddies!

Here, my draft (1st section), try to find out the mistakes!

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Proteins in endocytic pathway

Cells are not closed, independent units. Cells must undergo material exchange with their extracellular environment in order to function normally. Endocytosis is an essential process that allows the eukaryotic cells to take up materials from the outside. Here, the term endocytosis means the process by which cells absorb materials by engulfing it with cell membrane. By processing the endocyted materials intracellularly, cells are able to receive stimuli, information, nutrition, and control their membrane constituent. Since proteins often play roles in intercellular signalling and regulating cell function, the endocytosis of proteins is particularly crucial and sensitive.

Several endocytosis pathways have been discovered to date. However, the most widely utilised and best understood pathway is clathrin-mediated endocytosis. Large extracellular molecules such as proteins are absorbed mainly via this pathway though there are exceptions reported. In clathrin-mediated endocytosis, a clathrin coat will appear at inner curvature site. During this process, cell membrane buds inward to form small vesicles with clathrin coat, and both engulfed soluble materials and materials on the curvature membrane are internalised. The clathrin-mediated endocytosis is normally initiated by binding between transmembrane protein and intracellular clathrin. Therefore, soluble extracellular proteins are normally internalised together with their corresponding receptors which are able to bind clathrin via their intracellular domains. In this way, after vesicle budding, membrane bound receptors are on the vesicle membrane with their ligand in the vesicle lumen anchored on them.

The intracellular transportation routes of the proteins internalised by clathrin-mediated endocytosis are different. Nevertheless, several organelles – early endosomes (yeast counterpart post-Golgi endosomes, or PGEs), late endosomes (yeast counterpart prevacuole endosomes, or PVEs), multivesicular bodies (MVBs), lysosomes (in mammalian, with yeast counterpart names vacuoles) and Golgi apparatus – are the key points in their route. The internalised proteins are transiently stored, processed and/or sorted in these organelles. Among these organelles, Golgi apparatus is morphologically distinct and its main function is protein modification whilst the others are similar morphologically. Transportation of the internalised proteins among these organelles and cell surface is well controlled and is very complicated. Understanding of this field is important for advancing in cell biology. Here, I will concentrate on the intracellular sorting mechanism of membrane protein for degradation.

Early endosomes (PGEs), late endosomes (PVEs), MVBs and lysosomes (vacuoles)

Among the key organelles in internalised protein transportation route, the early endosomes, late endosomes, MVBs and lysosomes are all bilayer vacuolar structures. However, they have their distinct characteristics.

Early endosomes mainly distribute near plasma membrane, with irregular, tubovesicular shape. They have low osmium affinity, thus appears as light area under electron microscope. Early endosomes contains various kinds of proteins and has a typical pH around 6.0 which is lower than plasma pH (~7.2) and induces ligand-receptor disassociation. Early endosome is the location for first storage and sorting of internalised proteins. Rab7 is a specific protein marker for early endosomes. Late endosomes often have regular spherical or ellipsoid shape, and higher osmium affinity than early endosomes. Small vesicles can be found inside of late endosomes. Late endosomes mainly contain proteins for degradation and Rab5 is a specific protein marker for them. The term MVB is a morphologically defined group which stands for bilayer vacuole with multiple small vesicles inside. The MVBs are often very similar to late endosomes both morphologically and biochemically and may in fact be a portion of the late endosomes. Late endosomes with certain number of intralumental vesicles may be named MVBs. However, MVBs may also derive from early endosomes by budding. Lysosomes (vacuoles in yeast) are similar to late endosomes morphologically too, however, they have a pH lower than 5.0 and high level of acid hydrolases. The acid hydrolases activated under this pH degrade proteins within the lumen of lysosomes. Furthermore, lysosomes sometimes have multilamella structures inside, and are slightly more osmophobic than late endosomes.

It has been reported that late endosomes are matured from early endosomes. During this maturation process, morphology and protein comprising change continuously from early endosome to late endosome. The phenomenon of incolocalisation of some protein is both discovered in early endosomes and late endosomes recently. However, the mechanism of this phenomenon is unknown and no evidence of connection between this phenomenon and endosome maturation has been reported.

Protein transportation after internalisation

Generally, the clathrin coated vesicles formed in endocytosis are firstly fused with early endosomes and all internalised proteins are transported to early endosomes consequently. Ligand and receptors are disassembled and then initially sorted in the early endosome to be transported out to certain target or remain. Transportation of proteins among early endosomes is achieved by a tubular network which connects early endosomes together. Similar tubules are also reported to be intermediate for proteins moving from early endosomes to cell surface. Small vesicles, which are similar to the vesicles formed on plasma membrane during endocytosis, bud from and transport proteins among Golgi apparatus, early/late endosomes and extracellular environment. MVBs are structures which transport membrane proteins to lysosomes (or vacuoles in yeast). Transportation pathway between MVBs and endosomes/Golgi apparatus is still unclear. However, the proteins in/on MVBs may be inherited from early endosomes during maturation. Moreover, another organelle named exosomes which is very similar to MVBs morphologically and transport proteins to the extracellular space has been reported recently.

Depends on their function, proteins internalised have their determined fate such as degradation, storage, modification and recycling in the cells. Certain protein is transported to destined cell structures which are responsible for certain relevant processing step to meet their fate. Because of the important roles of some internalised proteins, correct intracellular delivery is crucial for the normal function of the cells. For this purpose, proper protein sorting is vital. Since most sorting events are occurred on endosomes, the endosomal sorting is a main field of study now.

Sorting of membrane protein for degradation

There are two main protein degradation units in eukaryote cells – proteasomes and lysosomes/vacuoles. As proteasomes are cytosomal complexes and are mainly responsible for cytosolic and nuclear protein degradation, membrane proteins destined for degradation are normally degraded in lysosomes/vacuoles [1, 2]. In time sequence, the intracellular localisation of these membrane proteins after internalisation is typically early endosomes, late endosomes/MVBs, and finally lysosomes/vacuoles. It is noticeable that they are presented on small vesicles in the lysosome/vacuole lumen before being degraded. The reason is that membrane proteins on these vesicles could be degraded by acid hydrolases in the lysosome/vacuole lumen but the membrane proteins on the limiting membrane of lysosome/vacuole could not. This membrane protein localisation is accomplished by a special sorting pathway.

As mentioned, internalised membrane proteins are presented on the membrane of clathrin coated vesicles. After fusion between early endosome and internalised vesicles, these proteins are transferred on early endosome membrane with their extracellular domain (before internalisation) facing lumenal space. The membrane proteins for degradation are consequently transported to MVB membranes with their extracellular domain facing lumenal space by unclear mechanisms as described. During the next step, these proteins are sorted to the small intralumenal vesicles (ILVs) within MVB. These ILVs are formed by inward budding of MVB limiting membrane and finally enter lysosome/vacuole by permanent (or possibly transient) fusion of MVB and lysosome/vacuole. This membrane protein sorting and ILV formation process is the last step before degradation, malfunction in this process will have fatal sequences and is thus strictly regulated.

It has been showed that the intracellular domains of most of the membrane proteins destined to lysosome/vacuole are monoubiquitinated before degradation and artificially fused monoubiquitinated membrane proteins can be efficiently transported to lysosome/vacuole. Monoubiquitin tag is hence considered as a common lysosome/vacuole degradation signal in cells. This also implies a single pathway rather than several protein specific pathways is responsible for sorting the monoubiquitinat tagged membrane proteins to the ILVs.

Proteins directly involves in this sorting were initially identified in yeast and are named Class E vacuolar protein sorting (Vps) proteins. Depletion of any single protein of these proteins will lead to morphologically aberrant MVBs, mislocalisation of proteins in MVBs and failure of membrane protein degradation. This phenotype caused by Class E Vps protein depletion is named “Class E phenotype” and the aberrant MVBs are named “Class E compartment” [3]. Later, conserved homologues of the yeast Class E Vps proteins were identified in other species, including mammalians. It has been discovered that a group of the Class E Vps proteins function on MVB membrane to directly sort membrane proteins into ILVs. These proteins are named endosomal sorting complex required for transport (ESCRT) proteins and the sorting pathway operated by them is named ESCRT pathway. Evidences so far have proved that all of the proteins sorted into ILVs within MVB are regulated by the ESCRT pathway.