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Cell membranes and transport hands on labs inc version

PDF Summary Traffic from the trans-Golgi network to the plasma membrane is thought to occur through at least two different independent pathways. The chitin synthase Chs3p requires the exomer complex and Arf1p to reach the bud neck of yeast cells in a cell-cycle-dependent manner, whereas the hexose transporter Hxt2p localizes over the entire plasma membrane independently of the exomer complex.

Here, we conducted a visual screen for communalities and differences between the exomer-dependent and exomer-independent transport to the plasma membrane in Saccharomyces cerevisiae. We found that most of the components that are required for the fusion of transport vesicles with the plasma membrane, are involved in localization of both Chs3p and Hxt2p.

However, the lethal giant larva homologue Sro7p is required primarily for the targeting of Chs3p, and not Hxt2p or other cargoes such as Itr1p, Cwp2p and Pma1p. Interestingly, this transport defect was more pronounced in large-budded cells just before cytokinesis than in small-budded cells.

In addition, we found that the yeast Rab11 homologue Ypt31p determines the residence time of Chs3p in the bud neck of small-budded, but not large-budded, cells. We propose that transport to and from the bud neck is regulated differently in small- and large-budded cells, and differs early and late in the cell cycle.

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Introduction Various transport pathways operate to localize cargo to the plasma membrane. At least two different transport pathways have been identified in yeast by the buoyant density of the transport carriers and the cargo they contain Gurunathan et al.

The high-density fraction transports exoglucanase and invertase, whereas the low-density pool brings the plasma membrane ATPase Pma1p, the glucose transporter Hxt2p, Fus1p and the chitin synthase III Chs3p, and probably a large variety of other cargo to the plasma membrane Bagnat and Simons, 2002 ; Barfield et al.

The lighter pool of transport containers can probably be further split into subclasses. Chs3p and Fus1p have distinct transport requirements from Pma1p or Hxt2p, because they need the action of the exomer complex for exit from the trans-Golgi network TGN Sanchatjate and Schekman, 2006 ; Trautwein et al.

They form complexes of varying stoichiometries and might act as receptors for cargoes to be transported by the exomer-dependent pathway Sanchatjate and Schekman, 2006 ; Trautwein et al.

Chs3p displays an interesting localization pattern as it changes over the cell-cycle. Early in the cell-cycle in G1 and S phase, Chs3p is located at the incipient bud site and at the bud neck of small-budded cells. In G2, cell membranes and transport hands on labs inc version the bud is medium sized, Chs3p is present in internal structures referred to as chitosomes Chuang and Schekman, 1996 ; Ziman et al. In mitosis, when also the actin cytoskeleton re-polarizes from the bud tip to the bud neck, Chs3p is exported to the bud neck again.

By contrast, the fusion of secretory transport vesicles is thought to occur at the incipient bud site and then in G1 and S phase of the cell cycle, at the bud tip. During G2, vesicles fuse all over the bud surface and subsequently, during cytokinesis, relocalization of the vesicle fusion site to the bud neck occurs. This raises the question whether fusion of Chs3p-containing transport vesicles is governed by the same regulatory factors as that of general transport vesicles.

It is assumed that the exocyst is required for fusion of transport vesicles with the plasma membrane because mutants in the exocyst component SEC6 accumulate vesicles TerBush et al. However, no detailed analysis is available. Here, we investigate how the transport of Chs3p to the bud neck region of the plasma membrane is controlled in Saccharomyces cerevisiae. We show that delivery of Chs3p to the bud neck region continues, even when the vesicle fusion machinery is only apparent in the bud tip.

To gain a better understanding of the regulation of Chs3p localization at the plasma membrane, we performed a visual screen comparing the transport of Chs3p and Hxt2p to the plasma membrane using fluorescently labeled variants.

We found that the general fusion machinery at the plasma membrane is essential for the efficient delivery of both cargoes to the plasma membrane, independent of whether the cargo is localized to the bud neck region or the entire plasma membrane.

However, some transport mutants affected the localization of Chs3p and Hxt2p, and other exomer-independent cargo differentially. Moreover, we found that the residence time of Chs3p at the bud neck is determined in different ways in small- and large-budded cells. Our data therefore indicate that general transport factors can selectively influence the temporal and spatial localization of different cargoes.

Results Transport dynamics of Chs3p and the exocyst complex over the yeast cell cycle To gain further insight into the temporal and spatial aspects of Chs3p transport, we observed localization of Chs3p—GFP over the cell cycle. As reported previously Valdivia et al. Transport vesicles generally fuse at the bud tip of small-budded cells, all over the bud surface in medium-sized cells and then vesicle fusion is restricted to the bud neck region at cytokinesis.

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The site of vesicle fusion is marked by the presence of the exocyst complex, which serves as tethering complex for vesicles with the plasma membrane Guo et al. We used Sec8p, a member of the exocyst complex TerBush and Novick, 1995fused to GFP to visualize the localization of the exocyst complex over the cell cycle Fig.

Sec8p—GFP was functional because a chromosomal fusion to the essential SEC8 was generated at the endogenous locus, and the resulting strain grew indistinguishably from an untagged control.

Interestingly, Chs3p—GFP remained restricted to the bud neck when the fusion machinery was localized to the bud tip.

This observation could be explained by two different possibilities. First, Chs3p—GFP transport is restricted to the short time-window of initiation of bud emergence. Second, Chs3p—GFP transport to the bud neck persists after bud emergence. To determine whether Chs3p could be delivered to the bud neck after the initial bud growth had been initiated, we performed a FRAP analysis. We bleached the Chs3p—GFP signal in small-budded cells and determined the reappearance of the Chs3p—GFP signal in the bud neck 10 minutes after the initial bleaching Fig.