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Vesicle Traffic and Organelle Inheritance
Summary: Susan Ferro-Novick is interested in how the specificity of vesicle traffic is maintained and how organelles are inherited from mother to daughter cells.
How organelles maintain their identity amid a constant flow of membrane traffic is an unanswered question in cell biology. Understanding how the specificity of vesicle traffic is achieved is key to addressing this question.
To ensure that a carrier vesicle reaches its correct final destination, vesicle coat proteins link cargo selection to vesicle tethering. 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 recruits the tether that physically links the transport vesicle to its appropriate acceptor compartment. Tethers also interact with Rab GTPases, molecular switches that regulate membrane traffic. Once the vesicle reaches its final destination, a SNARE on the vesicle pairs with its cognate SNARE on the target membrane. SNAREs are cytoplasmically oriented membrane proteins that mediate 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, the multiprotein tethering complex TRAPP (transport protein particle) is found in two forms: TRAPPI and TRAPPII. The two complexes share seven subunits, while three subunits are unique to TRAPPII. TRAPPI and TRAPPII are also multimeric guanine nucleotide exchange factors (GEFs) for the Rab GTPase Ypt1p. In yeast, this GTPase regulates endoplasmic reticulum (ER)-Golgi traffic and traffic from the early endosome to the late Golgi. Five of the seven subunits shared between TRAPPI and TRAPPII are essential for growth and are needed to fully activate Ypt1p. Mutational analysis revealed that the two TRAPP complexes mediate different transport steps. Mutations in TRAPPI subunits block ER-Golgi traffic, while mutations in TRAPPII-specific gene products block traffic from the early endosome to the late Golgi and within the Golgi. 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 a TRAPP subunit (Trs20).
The Role of Bet3 in Vesicle Tethering
Studies on vesicle traffic have focused on understanding how ER-derived vesicles (also called COPII vesicles) recognize and tether to their acceptor compartment. The budding of ER-derived vesicles depends on the COPII coat complex. The COPII coat is assembled when the GTPase Sar1p recruits the cargo adaptor complex Sec23p/Sec24p. This then leads to the capture of cargo by Sec24p, the recruitment of the Sec13p/Sec31p complex, and coat polymerization.
In mammalian cells, COPII vesicles first tether and fuse to each other (homotypic tethering/fusion) to form a pre-Golgi compartment. Cargo then moves from the pre-Golgi compartment to the Golgi. Mammalian Bet3 (mBet3) is enriched on the transitional ER (tER), the site where COPII vesicles are produced, and is required for homotypic COPII vesicle tethering in vitro. In small interfering RNA (siRNA)-treated mBet3-depleted cells, the architectures of pre-Golgi and Golgi compartments are disrupted, implying that mBet3 plays a pivotal role in the biogenesis of the Golgi.
In yeast, COPII vesicles do not tether and fuse with each other; instead, they fuse directly with the Golgi. Despite this difference, however, the mechanism of COPII vesicle tethering appears to be highly conserved. In higher and lower eukaryotes, the TRAPP subunit Bet3 mediates COPII vesicle tethering via an interaction with the coat subunit Sec23. This finding has led to the proposal that the binding of Bet3 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 play a role in determining the intracellular destination of a transport vesicle. Once binding occurs, the TRAPPI complex activates Ypt1p (Rab1 in mammalian cells), converting it from its GDP-bound to its GTP-bound form. This then leads to the recruitment of other tethers, such as Uso1p (p115 in mammals). The pairing of the SNAREs, which is a prerequisite to membrane fusion, is the final step in the docking and fusion of a COPII vesicle with acceptor membranes (see the figure).
The Mechanism of Ypt1p Activation by TRAPP
To address how TRAPP acts as a multimeric GEF, we crystallized Ypt1p bound to the minimal TRAPP subassembly required for GEF activity. This complex contains two copies of Bet3p (Bet3p-A and Bet3p-B) and one copy each of Trs23p, Bet5p, and Trs31p. The structure revealed that Ypt1p largely binds to the Trs23p subunit, although it also makes contact with the two copies of Bet3p and Bet5p. The carboxyl terminus of Bet3p-A induces a conformational change in the switch I region of Ypt1p, which leads to the release of GDP from Ypt1p. Bet5p links Bet3p-A to Trs23p, while Trs31p and Bet3p-B appear to facilitate the interaction of Ypt1p with Trs23p. This structure provides the first view of a multimeric tethering assembly complexed with the Rab it activates.
Organelle Inheritance
The ER is required for lipid synthesis and the proper folding and trafficking of proteins along the exocytic pathway. 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.
During cell division, organelles are duplicated and partitioned into daughter cells. Although the perinuclear ER appears to be connected to the cortical ER network, these two types of ER are delivered into daughter cells by different mechanisms. Our goal is to define the process by which cortical ER is delivered into daughter cells. To achieve this goal, we have used a genetic approach to identify the machinery that moves ER tubules from mother to daughter cells. In addition to identifying components of the ER inheritance machinery, this approach has identified two ER membrane proteins, Ice2p and Rtn1p, that play a role in the maintenance and structure of the cortical ER. Genetic screens have identified actin as the track and Myo4p as the motor that extends ER tubules into daughter cells. ER tubules bind to the exocyst at the bud tip of newly formed cells. The exocyst is a multisubunit complex that was originally identified for its role in tethering post-Golgi vesicles to the plasma membrane. 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 mitogen-activated protein (MAP) kinase Slt2p. We propose that the activation of Slt2p is required for the delivery of ER tubules to the bud tip. Experiments are currently in progress to address the role of Slt2p activation and inactivation in ER inheritance.
The organelle inheritance project is funded in part by a grant from the National Institutes of Health.
Last updated: November 5, 2008
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