Elizabeth Conibear

VPS35 Mutations in Parkinson Disease

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA‐II adaptors (found in plasma membrane coated pits) and the HA‐I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6‐phosphate (M6P) receptor‐a receptor which is concentrated in coated pits on both the plasma membrane and in the trans‐Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA‐II plasma membrane adaptor, which recognizes a ‘tyrosine’ signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA‐I adaptors. This suggests that there is an HA‐I recognition signal in another region of the M6P receptor tail, C‐terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans‐Golgi network, thus mediating efficient transfer of these enzymes to lysosomes.

Interaction landscape of membrane - protein complexes in Saccharomyces cerevisiae

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein–protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

Molecular Architecture and Functional Model of the Complete Yeast ESCRT-I Heterotetramer

The endosomal sorting complex required for transport-I (ESCRT-I) complex, which is conserved from yeast to humans, directs the lysosomal degradation of ubiquitinated transmembrane proteins and the budding of the HIV virus. Yeast ESCRT-I contains four subunits, Vps23, Vps28, Vps37, and Mvb12. The crystal structure of the heterotetrameric ESCRT-I complex reveals a highly asymmetric complex of 1:1:1:1 subunit stoichiometry. The core complex is nearly 18 nm long and consists of a headpiece attached to a 13 nm stalk. The stalk is important for cargo sorting by ESCRT-I and is proposed to serve as a spacer regulating the correct disposition of cargo and other ESCRT components. Hydrodynamic constraints and crystallographic structures were used to generate a model of intact ESCRT-I in solution. The results show how ESCRT-I uses a combination of a rigid stalk and flexible tethers to interact with lipids, cargo, and other ESCRT complexes over a span of approximately 25 nm.

An endocytic mechanism for haemoglobin‐iron acquisition in Candida albicans

The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem‐iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)‐anchored, haemin‐ and haemoglobin‐binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short‐lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C.  albicans vacuole. We relied on recapitulation of the Rbt51‐dependent haem‐iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin‐iron utilization and haemoglobin uptake. These mutants define a novel endocytosis‐mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway.

Palmitoylation by the DHHC protein Pfa4 regulates the ER exit of Chs3.

The yeast chitin synthase Chs3 provides a well-studied paradigm for polytopic membrane protein trafficking. In this study, high-throughput analysis of the yeast deletion collection identifies a requirement for Pfa4, which is an uncharacterized protein with protein acyl transferase (PAT) homology, in Chs3 transport. PATs, which are the enzymatic mediators of protein palmitoylation, have only recently been discovered, and few substrates have been identified. We find that Chs3 is palmitoylated and that this modification is Pfa4-dependent, indicating that Pfa4 is indeed a PAT. Chs3 palmitoylation is required for ER export, but not for interaction with its dedicated ER chaperone, Chs7. Nonetheless, both palmitoylation and chaperone association are required to prevent the accumulation of Chs3 in high–molecular mass aggregates at the ER. Our data indicate that palmitoylation is necessary for Chs3 to attain an export-competent conformation, and suggest the possibility of a more general role for palmitoylation in the ER quality control of polytopic membrane proteins.

Vacuolar biogenesis in yeast: sorting out the sorting proteins.

Institute of Molecular Biology University of Oregon Eugene, Oregon 97403-l 229 Roughly 10 years ago, a small number of yeast labs iso- lated a collection of mutants that missort the enzyme car- boxypeptidase Y (CPY) in an attempt to unravel vacuolar protein sorting, a process that is closely related to lyso- somal enzyme sorting in mammals. They came up with an overwhelming number of VfS genes-more than 50 so far-and the screen is not saturated (see Stack et al., 1995b). How can sense be made of such a large col- lection? Our current model of the vacuolar transport pathway is illustrated in Figure 1. The CPY receptor, together with its bound ligand, is sorted into vesicles that bud from the late Golgi and fuse with the prevacuole (step i), where the two dissociate, allowing the receptor to be recycled (step 2) while CPY is delivered to the vacuole in a distinct vesicle-mediated process (step 3). This pathway is not only required for vacuolar protein sorting, but is also important for maintaining the late Golgi distribution of a number of membrane proteins, which continuously leave the Golgi and are retrieved from the prevacuolar compartment by retrograde transport (reviewed by Nothwehr and Stevens, 1994). It takes a very large number of proteins to carry out just a single round of vesicle budding, targeting, and fusion, and every transport step requires a distinct (but related) set of proteins that confer specificity. The conservation of vesicle transport machinery at different transport steps and in different organisms has allowed observations drawn from yeast genetics, neuroscience, and biochemis- try to contribute to a general model of vesicle targeting and fusion known as the SNARE hypothesis (reviewed by Ferro-Novick and Jahn, 1994). In fact, a number of VPS genes encode proteins predicted to participate in the for- mation of SNARE complexes at several of the steps in Figure 1, thus accounting in part for the large number of VPS genes. Recent advances have focused considerable attention on an early step in vacuolar protein sorting: the budding of vesicles at the late Golgi that are targeted for the prevac- uole. Two particularly intriguing proteins have been impli- cated in the budding process, dynamin-like Vpsl p and the yeast lipid kinase Vps34p. Both of these proteins are highly related to mammalian proteins involved in the for- mation of clathrin-coated vesicles and are therefore likely to represent conserved elements in the budding machin- ery. By examining the role of Vpsl p and Vps34p in vacuo- lar protein sorting in yeast, we may gain considerable in- sight into general mechanisms of vesicle formation that operate in all eukaryotes.

Palmitoylation and depalmitoylation dynamics at a glance.

Protein palmitoylation, the thioester linkage of fatty acyl moieties (typically, saturated 16C palmitate) to cysteine, is a lipid modification that serves both to tether proteins to membranes and to direct their localization to membrane microdomains. Unlike the two other types of lipid modification

Studying yeast vacuoles

Publisher Summary This chapter describes methods for studying different aspects of vacuole biogenesis and function. It presents three different approaches for visualizing the yeast vacuole: staining with vital dyes, using green fluorescent protein (GFP)-tagged vacuolar marker proteins, and immunofluorescence microscopy of formaldehyde-fixed cells. Because protein targeting to the vacuole is central to its function, the chapter presents various methods for assessing the fidelity of protein sorting using soluble and membrane-bound hydrolases, such as marker proteins. The chapter also discusses various techniques for determining whether an uncharacterized protein is associated with and/or degraded in the vacuole. The use of stage-specific transport mutants to identify the route by which a particular protein reaches the vacuole is also examined in the chapter. The chapter concludes with a description of methods for purifying vacuoles based on their low buoyant density to carry out biochemical studies, such as the assembly and activity of the V-ATPase.

ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization

Dynamic changes in protein S-palmitoylation are critical for regulating protein localization and signaling. Only two enzymes - the acyl-protein thioesterases APT1 and APT2 – are known to catalyze palmitate removal from cytosolic cysteine residues. It is unclear if these enzymes act constitutively on all palmitoylated proteins, or if additional depalmitoylases exist. Using a dual pulse-chase strategy comparing palmitate and protein half-lives, we found knockdown or inhibition of APT1 and APT2 blocked depalmitoylation of Huntingtin, but did not affect palmitate turnover on postsynaptic density protein 95 (PSD95) or N-Ras. We used activity profiling to identify novel serine hydrolase targets of the APT1/2 inhibitor Palmostatin B, and discovered that a family of uncharacterized ABHD17 proteins can accelerate palmitate turnover on PSD95 and N-Ras. ABHD17 catalytic activity is required for N-Ras depalmitoylation and re-localization to internal cellular membranes. Our findings indicate that the family of depalmitoylation enzymes may be substantially broader than previously believed. DOI: http://dx.doi.org/10.7554/eLife.11306.001