The tectonic complex regulates membrane protein composition in the photoreceptor cilium.

The primary cilium is a signaling organelle with a unique membrane composition maintained by a diffusional barrier residing at the transition zone. Many transition zone proteins, such as the tectonic complex, are linked to preserving ciliary composition but the mechanism remains unknown. To understand tectonic's role, we generate a photoreceptor-specific Tctn1 knockout mouse. Loss of Tctn1 results in the absence of the entire tectonic complex and associated MKS proteins yet has minimal effects on the transition zone structure of rod photoreceptors. We find that the protein composition of the photoreceptor cilium is disrupted as non-resident membrane proteins accumulate in the cilium over time, ultimately resulting in photoreceptor degeneration. We further show that fluorescent rhodopsin moves faster through the transition zone in photoreceptors lacking tectonic, which suggests that the tectonic complex acts as a physical barrier to slow down membrane protein diffusion in the photoreceptor transition zone to ensure proper removal of non-resident membrane proteins.

Adaptor protein complex-1 (AP-1) is recruited by the HEATR5 protein Laa1 and its co-factor Laa2 in yeast.

Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat-containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized γ-ear-binding sites found in other AP-1-binding proteins. This motif in Laa2 is essential for the Laa1-AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the γ-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.

Investigation of Ldb19/Art1 localization and function at the late Golgi.

The arrestin-related family of proteins (ARTs) are potent regulators of membrane traffic at multiple cellular locations in the yeast Saccharomyces cerevisiae. Several ARTs act at multiple locations, suggesting that ARTs with well-established functions at one location may have additional, as of yet, uncharacterized roles at other locations in the cell. To more fully understand the spectrum of cellular functions regulated by ART proteins, we explored the localization and function of Ldb19/Art1, which has previously been shown to function at the plasma membrane, yet is reported to localize to the trans-Golgi network (TGN). We report that the C-terminal fusion of Ldb19 with GFP is functional and, as previously reported, localizes to the TGN. We further establish that Ldb19 associates with late stages of TGN maturation that are enriched in the clathrin adaptor protein complex-1 (AP-1). Additionally, we present genetic interaction assays that suggest Ldb19 acts at the late TGN in a mechanism related to that of AP-1. However, Ldb19 and AP-1 have dissimilar phenotypes in a subset of assays of membrane traffic, suggesting Ldb19 functions at the TGN are distinct from those of AP-1. Together these results indicate Ldb19 functions at the TGN, in addition to its well-established role in endocytosis.

A simple and inexpensive quantitative technique for determining chemical sensitivity in Saccharomyces cerevisiae.

Chemical sensitivity, growth inhibition in response to a chemical, is a powerful phenotype that can reveal insight into diverse cellular processes. Chemical sensitivity assays are used in nearly every model system, however the yeast Saccharomyces cerevisiae provides a particularly powerful platform for discovery and mechanistic insight from chemical sensitivity assays. Here we describe a simple and inexpensive approach to determine chemical sensitivity quantitatively in yeast in the form of half maximal inhibitory concentration (IC50) using common laboratory equipment. We demonstrate the utility of this method using chemicals commonly used to monitor changes in membrane traffic. When compared to traditional agar-based plating methods, this method is more sensitive and can detect defects not apparent using other protocols. Additionally, this method reduces the experimental protocol from five days to 18 hours for the toxic amino acid canavanine. Furthermore, this method provides reliable results using lower amounts of chemicals. Finally, this method is easily adapted to additional chemicals as demonstrated with an engineered system that activates the spindle assembly checkpoint in response to rapamycin with differing efficiencies. This approach provides researchers with a cost-effective method to perform chemical genetic profiling without specialized equipment.

Glucose starvation inhibits autophagy via vacuolar hydrolysis and induces plasma membrane internalization by down-regulating recycling.

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation.

High intensity and prevalence of two species of trematode metacercariae in the fathead minnow (Pimephales promelas) with no compromise of minnow anti-predator competence.

Opportunity for parasites to manipulate host behavioral phenotype may be influenced by several factors, including the host ecology and the presence of cohabiting parasites in the same host. Metacercariae of Ornithodiplostomum ptychocheilus and "black spot" Crassiphiala bulboglossa have similar life cycles. Each parasite uses a littoral snail as a first intermediate host, fathead minnows as a second intermediate host, and a piscivorous bird as a final host. Metacercariae of black spot encyst in the dermal and epidermal tissues, while metacercariae of O. ptychocheilus encyst on the brain over a region that coordinates optomotor responses. Because of site differences within the host, we predicted that O. ptychocheilus metacercariae might manipulate the behavioral phenotype of minnows to facilitate transmission to the final host, but metacercariae of black spot would not. In our study population, prevalence was 100% for O. ptychocheilus , with an overall median intensity of 105 metacercariae per minnow. Prevalence of black spot was 60%, with a median abundance and intensity of 12 and 20 metacercariae per minnow for the overall sample and for infected fish, respectively. Minnows accumulated both parasites over time, producing significant correlations between intensity and minnow body length and between intensities of the 2 parasites. Minnows infected with black spot had on average twice as many O. ptychocheilus metacercariae as similar-sized minnows without any black spot cercariae. We found no correlation between body condition of minnows and intensity for either parasite. We measured 2 aspects of anti-predator competence to test for effects linked to parasite intensity. We found no correlation between intensity of either species of parasite and latency to behavioral response to attack from a mechanical model heron, nor was there any effect of parasite intensity on a measure of shoaling affinity. The absence of any detectable effect of metacercariae on anti-predator competence in minnows may reflect selection against parasite pathology from predation by non-hosts of the parasites and overwinter mortality due to low dissolved oxygen.