How organelles maintain their identity amid a constant flow of membrane traffic has been an unanswered question in cell biology. Understanding how the specificity of vesicle traffic is achieved is key to addressing this question. Our findings indicate that coat proteins link cargo selection to vesicle tethering to ensure that a carrier vesicle reaches its correct final destination. The coat, which is released at the target membrane, also prevents premature fusion of the vesicle. Vesicle coat complexes are composed of two layers, an inner core that sorts cargo (coat adaptor complex) and an outer shell that deforms the membrane to form a vesicle. The inner core of the coat also recruits the machinery that tethers the transport vesicle to its appropriate acceptor compartment. Tethers interact with Rab GTPases, molecular switches that regulate membrane traffic. Once the vesicle reaches its final destination, a SNARE on the vesicle pairs, via its “SNARE motif,” with its cognate SNARE on the target membrane. SNAREs are cytoplasmically oriented membrane proteins that catalyze membrane fusion. Thus, the specificity of membrane traffic appears to be ensured by the concerted action of multiple players: coat proteins, tethers, Rabs, and SNAREs. Each of these players adds a level of specificity to vesicle traffic. This ultimately guarantees that a carrier vesicle only fuses with its correct acceptor compartment.
TRAPP and a Human Disease
In the yeast Saccharomyces cerevisiae, there are three forms of the TRAPP (transport protein particle) complex. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) for the Rab GTPase Ypt1. These complexes share six subunits (Figure 1). The smallest complex, TRAPPI, only contains the core subunits. The largest complex, TRAPPII, contains the six core subunits plus three additional subunits (Trs130, Trs120 and Trs65), while TRAPPIII contains the six core subunits and one unique subunit (Trs85). TRAPP subunits are highly conserved from yeast to man. Interestingly, spondyloepiphyseal dysplasia tarda, a recessive disorder in bone formation, is caused by mutations in the human ortholog of Trs20, a TRAPP subunit that is present in all three complexes.
Ypt1 and its mammalian homologue Rab1 regulate endoplasmic reticulum (ER)-Golgi traffic, Golgi traffic and autophagy. TRAPPII-and TRAPPIII-specific subunits target the TRAPP complexes to different cellular locations where, in combination with other factors, they mediate a specific membrane tethering event. Mutations in TRAPPI gene products block ER-Golgi traffic, while mutations in TRAPPII-specific gene products block Golgi traffic. The loss of the TRAPPIII-specific subunit Trs85 leads to a defect in autophagy in both yeast and mammalian cells.
Macroautophagy, a highly conserved catabolic process by which organelles and proteins are delivered to the vacuole/lysosome for degradation, is induced by a variety of physiological cues. Defects in this process have been linked to neurodegerative diseases such as Parkinson’s disease and cancer. During macroautophagy, 30-60 nm vesicles (called Atg9 vesicles) tether and fuse to form the phagophore, a cup-shaped membrane that expands to form an autophagosome, a compartment that delivers organelles and proteins to the vacuole/lysosome for degradation. The TRAPPIII complex, Ypt1/Rab1 and its effector the Atg1 kinase have been implicated in tethering Atg9 vesicles.
The Role of the COPII Coat in Vesicle Tethering
The different TRAPP complexes all appear to participate in different membrane tethering events. The best understood of these tethering events is the tethering of ER-derived vesicles (also called COPII vesicles) with their acceptor compartment. The budding of ER-derived vesicles depends on the COPII coat complex. The COPII coat is assembled when the GTPase Sar1 recruits the cargo adaptor complex Sec23/Sec24. This leads to the capture of cargo by Sec24. Sec24 sorts a variety of cargo, including the SNAREs, into the vesicle. The recruitment of the coat outer shell, the Sec13/Sec31 complex, leads to coat polymerization and vesicle budding.
In mammalian cells, COPII vesicles first tether and fuse to each other to form a pre-Golgi compartment. Cargo then moves from the pre-Golgi compartment to the Golgi. In yeast, however, COPII vesicles fuse directly with the Golgi. Despite this difference, the mechanism of COPII vesicle tethering appears to be highly conserved. In both higher and lower eukaryotes, the most conserved TRAPP subunit Bet3 mediates COPII vesicle tethering via an interaction with the coat subunit Sec23. Our finding that the Bet3/Sec23 interaction is required for vesicle tethering led to the proposal that the binding of the TRAPPI complex to Sec23 marks a coated vesicle for fusion with another coated vesicle or the Golgi. It also implies that the coat and its associated cargo plays a role in determining the intracellular destination of a transport vesicle.
We found that TRAPPI can only bind to Sec23 after Sar1 has been released from the vesicle. As Sar1 is required for vesicle fission, this finding implies that vesicle fission must be completed before TRAPPI initiates vesicle tethering (Figure 2, step 1). TRAPPI recruits and activates Ypt1/Rab1 (step 2), which subsequently binds to its effector Uso1 (p115 in mammalian cells) (step 3). Uso1 is a long coiled-coil tether that links the vesicle to the Golgi (4). Once the vesicle tethers to the Golgi, the serine/threonine kinase Hrr25 (CKIδ in mammalian cells) displaces the TRAPPI complex from Sec23 and phosphorylates the COPII coat (step 4). Phosphorylation of the coat cycles it off membranes, and the release of the coat from the vesicle allows the SNAREs to pair (step 5). Thus, through sequential interactions with three different binding partners (Sar1, TRAPPI and Hrr25), the coat subunit Sec23 mediates the direction of membrane flow and ensures that the COPII coat will only be released once the vesicle reaches its target membrane. The serine/threonine phosphatase Sit4 (PP6 in mammals) dephosphorylates the coat to allow for a new round of vesicle budding (step 6). These events appear to be conserved from yeast to man.
ER Stucture and Inheritance
The ER is required for lipid synthesis, Ca2+ storage and the proper folding and trafficking of proteins destined for export. In budding yeast, the ER has been divided into classes: the tubules that line the plasma membrane are referred to as cortical or peripheral ER, while the tubular connections to the nuclear envelope are called perinuclear ER. As in mammalian cells, the cortical ER forms a dynamic network of interconnected tubules.
Our goal is to define the process by which the cortical ER achieves its unique structure and the mechanism by which it is delivered into daughter cells. To achieve this goal, a genetic screen has been used to identify mutants with altered structure or defective inheritance. These screens have identified actin as the track and Myo4p as the motor that extends ER tubules into daughter cells. Once the ER arrives at the bud tip it is retained in the daughter cell and then distributed along the periphery of the bud as it grows. We have shown that Ptc1p, a serine/threonine phosphatase, regulates this event by modulating the activity of the pool of mitogen-activated protein (MAP) kinase Slt2 at the bud tip.
In addition to the components of the ER inheritance machinery, several membrane proteins play a role in the maintenance and structure of the cortical ER. The conserved reticulon protein Rtn1 works with Yop1 (DP1 in mammalian cells) to shape ER tubules, while Atlastin/Sey1 mediates ER-ER tubule fusion. Lnp1 localizes to the three-way junctions of the ER and works synergistically with the reticulons and Yop1, but in antagonism to Atlastin/Sey1 to maintain the ER network. Mutations in DP1 family members and Atlastin have been associated with certain neurological diseases.
The organelle inheritance project has been funded in part by a grant from the National Institutes of Health.
As of November 18, 2013