The exocyst -- an octameric protein complex mediating vesicle tethering at the plasma membrane for exocytosis -- is a downstream effector of the Rab proteins Rab8 and Rab11, which are key regulators of membrane trafficking from the trans-Golgi network and recycling endosome to the plasma membrane. Rab11 and Rab8 coordinate their actions via Rabin8, the guanine nucleotide exchange factor of Rab8. A cascade of protein-protein interactions involving the Rabs and the exocyst complex couples the generation of secretory vesicles at donor compartments to their docking and fusion at the plasma membrane. Here, we discuss recent work implicating Rab proteins and the exocyst in primary ciliogenesis and epithelial lumenogenesis. In addition, we discuss early work in the budding yeast Saccharomyces cerevisiae, which provided initial insight into the molecular mechanisms of polarized exocytosis.

The Gal4-UAS system was used to overexpress or knock down β spectrin with dsRNA in a variety of Drosophila tissues. Unexpectedly, overexpression in most tissues tested was lethal, whereas knockdown failed to produce a detectable phenotype in the same tissues. The lethality of a β spectrin mutation was rescued by expression of β spectrin in neurons.

The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential β spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a β spectrin transgene or to knock down β spectrin expression with dsRNA. The results show that 1) overexpression of β spectrin in most of the cell types studied was lethal; 2) knockdown of β spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-β spectrin transgene was sufficient to overcome the lethality of a loss-of-function β spectrin mutation. Thus β spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for β spectrin function in vivo.

Background

Current models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1) was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE) as a marker for studies of the downstream effects of spectrin cytoskeleton mutations.

Results

Sequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila.

Conclusions

Based on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

Prevailing models place spectrin downstream of ankyrin in a pathway of assembly and function in polarized cells. We used a transgene rescue strategy in Drosophila melanogaster to test contributions of four specific functional sites in β spectrin to its assembly and function. (1) Removal of the pleckstrin homology domain blocked polarized spectrin assembly in midgut epithelial cells and was usually lethal. (2) A point mutation in the tetramer formation site, modeled after a hereditary elliptocytosis mutation in human erythrocyte spectrin, had no detectable effect on function. (3) Replacement of repetitive segments 4–11 of β spectrin with repeats 2–9 of α spectrin abolished function but did not prevent polarized assembly. (4) Removal of the putative ankyrin-binding site had an unexpectedly mild phenotype with no detectable effect on spectrin targeting to the plasma membrane. The results suggest an alternate pathway in which spectrin directs ankyrin assembly and in which some important functions of spectrin are independent of ankyrin.