Introduction

Movement of proteins and other molecules through the endocytic and secretory pathways requires that material is continually shuttled to and from the plasma membrane and between organelles. These ongoing processes are mediated by a specialised set of miniature membrane enclosed 'containers' called transport vesicles which package material up, bud off from the organelle membrane and transport the enclosed cargo to its correct location.

With such a large number of distinct membrane enclosed organelles within the cell and a seemingly never ending flow of membrane traffic, vesicular transport is tightly regulated to ensure that vesicles reach and fuse with the relevant cellular compartment. A number of specialised proteins and lipids associated with vesicle and organelle membranes co-operate to help achieve this. Some of the key classes of proteins and lipids which ensure the specificity of vesicular transport are Rab GTPases and Phosphoinositides:

 

Rab GTPases

  • Rabs are small monomeric GTPases which are key regulators and co-ordinators of membrane traffic.
  • Over 60 different types of Rabs exist in mammals. Each one is often found associated with a specific organelle membrane following their activation, helping to define the identity of each organelle and provide a 'landmark' of sorts for vesicles in transit. 
  • As GTPases, Rabs switch between active and inactive GTP and GDP bound forms. In their inactive GDP bound form Rabs exist in cytosolic pools. They are activated by a specific guanine nucleotide exchange factor (GEF) which catalyses the exchange of GDP for GTP, resulting in association of Rabs with their target organelle membrane.  
  • Following activation Rabs recruit multiple effector proteins to their location. Effectors include sorting adaptors, tethering proteins, kinases, phosphatases, motor proteins and SNARE proteins. These are involved in almost all stages of vesicular transport and help to perform the specialised reactions required at each step (their roles are discussed in greater detail below). 
  • Rab cascades and crosstalk between GTPases allows sequential activation of Rabs required further along the vesicular transport pathway and co-ordination of Rab function, helping to guide vesicles toward their destination and recruit the right effectors at the right time to do so.
  • Having performed their function, GTPase activating proteins (GAP's) inactivate Rabs by facilitating hydrolysis of GTP to GDP, resulting in their removal from the membrane.

 

Phosphoinositides

  • Phosphoinositides (PI's) are lipids synthesised at the ER which can be modified by the addition of a phosphate group at the 3', 4' or 5' position of their lipid head group, producing 7 different phosphoinositide species in vivo.
  • Like Rabs, the subcellular distribution of phosphoinositides contributes to defining organelle identity and different species of phosphoinositide can be found localised to a subset of specific organelle membranes (figure 1 shows the known intracellular localisation of phosphoinositides and GTPases). This distribution of phosphoinositides on the cells membrane system is maintained by phosphoinositide specific kinases and phosphatases which are spatially restricted within the cell. To help this, PI kinases and phosphatases are often paired with specific Rabs and act as Rab effectors. The ease with which PI's can be rapidly inter-converted provides the cell with a very easy way to maintain organelle identity.
  • Different species of phosphoinositide at their respective organelle membranes can bind differing Rab GTPases, GEF's, GAP's and effectors. Phosphoinositide binding modules present in these proteins recognise certain lipid head groups displayed by different phosphoinositide species. PI's can thus work synergistically with Rabs to both define organelle identity and regulate membrane traffic, as is described below in the examples of membrane microdomain formation and recruitment of coat components during vesicle formation. 

 

    Further organisation of Rabs and Phosphoinositides: Membrane microdomains

    Positive feedback loops involving PI's, PI kinases, Rab GTPases, GEF's and effectors promote the formation of functionally distinct microdomains on membranes. On the same organelle membrane, microdomains enriched in different Rab proteins are often found; for instance, on the early endosome, Rab4 and Rab5 can be found in separate well defined regions apart from one another. Concentrating related phosphoinositides, Rabs and effectors within microdomains allows vesicles budding from this region to be 'pre-packaged' with all the equipment required to set them en route to their target destination and incoming vesicles of a simliar makeup to be confined to this specialised domain on arrival. Organelles can thus efficiently organise membrane traffic from disparate cellular locations and of varying composition within specific regions on its own membrane. 

    FIgure 1 - Organelle specific localisation of GTPases and PI's defines organelle identity



    The process of vesicular transport

    The process of vesicular transport can be broken down into three key steps:

    1. Vesicle formation and budding
    2. Vesicle transport and tethering
    3. Vesicle fusion with the target membrane

    Cargo passing through the endocytic and secretory pathways can often undergo successive rounds of budding and fusion until reaching its final destination. The key steps involved are described below in detail and illustrated in a more simplified form in figures 2 & 3.

    1. Vesicle formation and budding

    • Vesicle formation begins with the recruitment of specialised coat proteins and sorting adaptors which are involved in cargo selection and vesicle budding. 
    • Coat proteins are recruited to the membrane through the action of coat recruitment GTPases such as Arf or Sar1. These act in a similar way to Rab GTPases, embedding in the membrane following GTP binding before recruiting effectors (in this case, coat components).
    • There are three main types of coat protein which are involved in transport to and from differing cellular compartments. These are summarised in table 1: 

    Table 1 - Types of coat protein involved in vesicular transport



      • Cargo is selected and incorporated into budding vesicles through sorting signals present in its amino acid sequence which are in turn recognised by specific consensus sequences present in coat proteins and sorting adaptors. 
      • In the case of COPI and COPII coated vesicles, cargo binds directly to these coat proteins. Cargo to be packaged into clathrin coated vesicles however, binds to clathrin indirectly through the use of sorting adaptors, of which there are there are several different types within cells. The use of numerous different coat proteins and sorting adaptors allows cells to recognise and select different cargo and package it into vesicles destined for the right location.
      • Phosphoinositides also play a role in coat recruitment as they can bind coat components directly, stabilising their association with the membrane.
      • Assembly of coat proteins on the donor organelle membrane initiates the budding process by deforming the membrane region containing the cargo to be transported. Coat proteins join together and polymerise, giving shape to the vesicle by forming a cage around it.
      • Once the coated bud has matured further, accessory proteins such as Dynamin mediate vesicle scission by wrapping around the neck of the budding vesicle and helping it to pinch off from the organelle membrane.

          Figure 2 - Vesicle formation and budding



          2. Vesicle transport and tethering

          • Coat proteins can hinder vesicle fusion and having left the donor organelle, vesicles must quickly shed their coat. Hydrolysis of coat recruitment GTPases to their GDP bound form and Rab protein recruitment of PI phosphatases (which remove a phosphate group from membrane bound PI's involved in coat recruitment) help the vesicle to shed its coat.
          • Depending on the vesicles destination, further activated Rabs recruit specific motor proteins such as Myosin, Dynein or Kinesin which associate with microtubules or actin filaments (cytoskeletal filaments) and guide the vesicle towards its target.
          • The vesicle is then captured by a large multi-subunit complex called a tethering protein, recruited by a Rab on the acceptor organelle membrane, which positions the vesicle over the relevant microdomain ready for fusion. The presence of more than one Rab binding site on tethering complexes allows cross-linking between Rabs on the incoming vesicle and the acceptor organelle membrane over long distances.
          • Tethers are also important for the next transport step as they contribute to linking vesicle tethering to membrane fusion.

          3. Vesicle fusion with the target membrane

          • The final step in vesicular transport involves docking and fusion of the captured transport vesicle with the acceptor organelle membrane.
          • Fusion of the two membranes is mediated by complementary SNARE proteins on the transport vesicle and acceptor organelle membrane. Transport vesicles contain v-SNAREs comprised of a single polypeptide, whilst the acceptor organelle membrane contains a corresponding t-SNARE composed of 2-3 polypeptides.
          • When brought into close enough proximity v and t-SNAREs intertwine, overcoming the energy requirements which pose a boundary to membrane fusion. The two opposing bilayers come together and merge seamlessly, with the vesicle membrane integrating with the acceptor organelle membrane. Soluble cargo bound to cargo receptors on the vesicle is released into the organelle / extracellular space.
          • SNARE's provide another layer of specificity in membrane transport. Specific pairs have been shown to be involved in defined transport steps (i.e. ER to Golgi transport), therefore preventing fusion of vesicles with the wrong organelle. Further specificity is also thought to be provided by interactions of SNAREs with Rabs and accessory proteins which regulate SNARE activity. 

           

          Figure 3 - Vesicle transport, tethering and fusion



          Vesicular transport and disease

          Defects in components involved in vesicular transport are responsible for a number of human disease states. Rab GTPases have been implicated in cancer. Rab25 is overexpressed in many breast and ovarian cancers and correlates with poor survival rates - this is potentially linked to an aberrant involvement of overexpressed Rab25 in trafficking of membrane receptors through early endosomes. Numerous inherited diseases are also caused by mutations in vesicular transport machinery. Mutations in the Rab27, Rab27A and myosin VA genes can result in a form of partial albinism known as Griscelli syndrome. All of these gene products are in some way involved in the movement of melanosomes (skin pigment granules) towards the periphery of melanocytes (skin pigment producing cells) illustrating how defects in the trafficking of cellular cargo can have pathological consequences.

          References

          These reviews and textbooks explain some of the key the concepts of vesicular trafficking in greater detail:

          Alberts, B et al. Molecular Biology of the Cell, 5th Edition (2008). Chapter 13: pages 754-64. Garland Science.

          Behnia, R and Munro, S (2005). "Organelle identity and the signposts for membrane traffic." Nature. Dec 1; 438 (7068): 597-604.

          Hong, W (2005). "SNAREs and traffic." Biochim Biophys Acta. Jun 30; 1744(2): 120-44.

          Jahn, R and Scheller, R.H. (2006). "SNAREs - engines for membrane fusion." Nat. Rev. Mol Cell Biol. Sep; 7(9): 631-643.

          Paolo, G.D. and De Camilli, P (2006). "Phosphoinositides in cell regulation and membrane dynamics." Nature. Oct 12; 443 (7112): 651-7.

          Stenmark, H (2009). "Rab GTPases as coordinators of vesicle traffic." Nat. Rev. Mol. Cell Biol. Aug (8) 10: 513-25.

           

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