Expression of regions of Apm1p in E coli. and purification of the expressed proteins [abstract]

Abstract only availableAn active area of cell biological research is the study of intracellular protein trafficking. Improper function of protein trafficking has pathological consequences, as the malfunction of certain transport systems can give rise to certain cancers. Our lab focuses on the dynamic mechanisms of transport between the trans-Golgi network (TGN) and endosomes using the model protein Ste13p. The goal of my project is to deepen our understanding of Ste13p retrieval from the early endosome to the TGN via binding of the adaptor complex (AP-1). Previous research suggests that the C-terminal domain of the AP-1 subunit Apm1p interacts with a sorting signal contained within amino acids 1-12 of the cytosolic domain of Ste13p. With this in mind, we developed plasmids that expressed different regions of the C-terminal domain of Apm1p in E coli. The hope was to find a construct that provided both high expression and ease of purification. As part of a collaboration, we intended to discover how the 1-12 region of Ste13p binds to Apm1p on an atomic level. Each construct was contained within the expression vector pET28a(+) which is under the control of a T7 promoter. The results led us to choose Apm1p 161-475 as the best construct. Using a nickel column, we purified this Apm1p construct due to the nickel's binding affinity for the engineered 6xHistidine tag. Soon, we will perform a pull down assay to confirm the binding of Apm1 161-475 to a peptide that represents amino acids 1-12 of Ste13- GST. After completion of the assay, we will send our construct to our collaborator who will co-crystallize the AP-1 derivative with a Ste13p peptide to structurally define the binding between these two proteins. Structural data will make it easier to understand how mutations could affect the binding of the signal with the adaptor protein, thus allowing more detailed manipulations of both proteins

A search for regulators of a yeast synaptojanin

Abstract only availableThe yeast S. cerevisiae expresses three synaptojanins: Inp51p, Inp52p, and Inp53p. These enzymes are characterized by two specific characteristics. They contain an inositol 5'-phosphatase and a polyphosphoinositide phosphatase. Together, these enzymes play a crucial role in the membrane trafficking of yeast cells. The synaptojanins are important to yeast because the cells ability to survive is dependent on them. It has been shown that a complete knockout of the three genes causes lethality in yeast. The synaptojanin that we are focusing on in this experiment is Inp53p. Inp53p is involved in intra-cellular membrane trafficking within yeast. Loss of Inp53p function results in quicker membrane protein movement towards the prevacuolar compartment from the trans-Golgi network. Although it is known that these enzymes are regulated, the method of regulation is unknown. We wish to identify proteins in yeast that activate Inp53p. At the current time, we are constructing a strain of yeast that codes for a knockout of all three synaptojanins, which would have lethal results, and introducing a gene containing the INP53 gene fused to a weak promoter from GAL4 resulting in lower than normal expression of Inp53p. At the current time, no results have been recorded. However, in the near future we will transform the created strain with a gene library (YEp351) and look for proteins that, when over expressed, enhance the activity of Inp53p. On a broader scale, due to the large responsibility of synaptojanic activity in membrane trafficking in humans, any further research in the field would be beneficial to human health.Life Sciences Undergraduate Research Opportunity Progra

An in vitro binding assay to identify amino acid residues in Ste13p necessary for Ste13p/Amp1p interaction [abstract]

Abstract only availableCertain proteins in eukaryotic cells, such as the yeast Saccharomyces cerevisiae, are compartmentalized into intracellular organelles through trafficking among the trans-golgi network (TGN), the early endosome (EE) and the late endosome (LE). Some of these proteins are then transported to the vacuole. Ste13p, an enzyme that processes the mating pheromone -factor in yeast, moves through from the TGN to the EE and LE and back again. The retrieval of Ste13p from the EE is facilitated by Apm1p, a component of the AP-1 adaptor complex. It has been shown that Apm1p interacts with residues 1-12 of the amino terminus of Ste13p when the residues are fused to glutatione-s-transferase (GST). My project aims to determine which of these residues are essential for this interaction. In order to do this I created constructs with different deletions in this region of the gene for Ste13 in the context of a Ste13-GST fusion construct. I transformed these constructs into E. coli, expressed the Ste13-GST protein, and attached these proteins to gluthatione agarose beads. I will use these beads to perform pull down assays with purified Apm1p tagged with 6xHis. If the Apm1p is pulled down by the Ste13-GST beads but not by the GST alone, this will indicate that it associates with Ste13p. Mutant derivatives of Ste13-GST will be tested in the same fashion. Through analysis of the different mutants, we will be able to determine which amino acids are critical for this association, and by extension, critical for Ste13p retrieval from the EE back to the TGN

The clathrin adaptor complex 1 directly binds to a sorting signal in Ste13p to reduce the rate of its trafficking to the late endosome of yeast

Yeast trans-Golgi network (TGN) membrane proteins maintain steady-state localization by constantly cycling to and from endosomes. In this study, we examined the trafficking itinerary and molecular requirements for delivery of a model TGN protein A(F→A)–alkaline phosphatase (ALP) to the prevacuolar/endosomal compartment (PVC). A(F→A)-ALP was found to reach the PVC via early endosomes (EEs) with a half-time of ∼60 min. Delivery of A(F→A)-ALP to the PVC was not dependent on either the GGA or adaptor protein 1 (AP-1) type of clathrin adaptors, which are thought to function in TGN to PVC and TGN to EE transport, respectively. Surprisingly, in cells lacking the function of both GGA and AP-1 adaptors, A(F→A)-ALP transport to the PVC was dramatically accelerated. A 12-residue cytosolic domain motif of A(F→A)-ALP was found to mediate direct binding to AP-1 and was sufficient to slow TGN→EE→PVC trafficking. These results suggest a model in which this novel sorting signal targets A(F→A)-ALP into clathrin/AP-1 vesicles at the EE for retrieval back to the TGN

Structural Features of Vps35p Involved in Interaction with Other Subunits of the Retromer Complex

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73789/1/j.1600-0854.2007.00659.x.pd

The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network

Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP

Development and validation of the Self-Regulation of Eating Behaviour Questionnaire for adults

Background Eating self-regulatory capacity can help individuals to cope with the obesogenic environment and achieve, as well as maintain, a healthy weight and diet. At present, there is no comprehensive, reliable and valid questionnaire for assessing this capacity and measuring change in response to self-regulation interventions in adults. This paper reports the development of the Self-regulation of Eating Behaviour Questionnaire (SREBQ) for use in UK adults, and presents evidence for its reliability and construct validity. The development of the SREBQ involved generation of an item pool, followed by two pilot studies (Samples 1 and 2) and a test of the questionnaire’s underlying factor structure (Sample 3). The final version of the SREBQ was then assessed for reliability and construct validity (Sample 4). Results Development of the SREBQ resulted in a 5-item questionnaire. The face validity was satisfactory, as assessed by the pilot studies. The factor structure analysis (Sample 3) suggested that it has a single underlying factor, which was confirmed in a second sample (Sample 4). The SREBQ had strong construct validity, showing a positive correlation with general measures of self-regulation. It was also positively correlated with motivation and behavioural automaticity, and negatively correlated with food responsiveness and emotional over-eating (p < 0.001). It showed good discriminant validity, as it was only weakly associated with satiety responsiveness, food fussiness and slowness in eating. Conclusions The SREBQ is a reliable and valid measure for assessment of eating self-regulatory capacity in the general UK adult population

Genetic interactions between a phospholipase A2 and the Rim101 pathway components in S. cerevisiae reveal a role for this pathway in response to changes in membrane composition and shape

Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A2 (PLA2s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA2 in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA2 activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA2 activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them