Jia-Jia Liu

Support for the Prion Hypothesis for Inheritance of a Phenotypic Trait in Yeast

A cytoplasmically inherited genetic element in yeast, [PSI+], was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35—green fluorescent protein fusion. Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did [PSI+] inheritance. The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of [PSI+]. [PSI+] altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

Oligopeptide-repeat expansions modulate ‘protein-only’ inheritance in yeast

The yeast [PSI +] element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a ‘protein only’ mechanism reminiscent of the ‘protein-only’ transmission of mammalian prion diseases,. The underlying molecular mechanisms for both are poorly understood and it isnot clear how similar they might be. Sup35, the [PSI +] protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats—PQGGYQQYN in Sup35 and PHGGGWGQ in PrP,. Repeat expansions in PrP produce spontaneous prion diseases,. Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI +] elements, the first mutation of its type among these newly described elements of inheritance. In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in β-sheets and form higher-order structures much more rapidly than wild-type peptides. Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.

Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion [PSI+]

The yeast prion [PSI+] provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation. [PSI+] is a nonfunctional, ordered aggregate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state. The N-terminal region of Sup35p plays a central role in prion induction and propagation. The C-terminal region provides translation termination activity. The function of the highly charged, conformationally flexible middle region (M) is unknown. An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble. Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable. Substituting charged flexible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type [PSI+] elements), but had profoundly different properties. One was soluble in both the prion and the nonprion form, mitotically stable but meiotically unstable, and cured by guanidine HCl but not by alterations in heat shock protein 104 (Hsp104p). The other could only maintain the prion state in the presence of wild-type protein, producing Mendelian segregation patterns. The unique character of these M variants, all carrying the same N-terminal prion-determining region, demonstrate the importance of M for [PSI+] and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prion-like changes in protein conformation than suggested by our current methods for defining prion states.

Microtubule-associated protein 1B: a neuronal binding partner for gigaxonin

Giant axonal neuropathy (GAN), an autosomal recessive disorder caused by mutations in GAN, is characterized cytopathologically by cytoskeletal abnormality. Based on its sequence, gigaxonin contains an NH2-terminal BTB domain followed by six kelch repeats, which are believed to be important for protein–protein interactions (Adams, J., R. Kelso, and L. Cooley. 2000. Trends Cell Biol. 10:17–24.). Here, we report the identification of a neuronal binding partner of gigaxonin. Results obtained from yeast two-hybrid screening, cotransfections, and coimmunoprecipitations demonstrate that gigaxonin binds directly to microtubule-associated protein (MAP)1B light chain (LC; MAP1B-LC), a protein involved in maintaining the integrity of cytoskeletal structures and promoting neuronal stability. Studies using double immunofluorescent microscopy and ultrastructural analysis revealed physiological colocalization of gigaxonin with MAP1B in neurons. Furthermore, in transfected cells the specific interaction of gigaxonin with MAP1B is shown to enhance the microtubule stability required for axonal transport over long distance. At least two different mutations identified in GAN patients (Bomont, P., L. Cavalier, F. Blondeau, C. Ben Hamida, S. Belal, M. Tazir, E. Demir, H. Topaloglu, R. Korinthenberg, B. Tuysuz, et al. 2000. Nat. Genet. 26:370–374.) lead to loss of gigaxonin–MAP1B-LC interaction. The devastating axonal degeneration and neuronal death found in GAN patients point to the importance of gigaxonin for neuronal survival. Our findings may provide important insights into the pathogenesis of neurodegenerative disorders related to cytoskeletal abnormalities.

The retromer component SNX6 interacts with dynactin p150 Glued and mediates endosome-to-TGN transport

The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network (TGN). It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins (SNXs). Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2s dimerization partners, SNX6, interacts with the p150Glued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-p150Glued interaction. Disruption of the SNX6-p150Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.

A Functional Dynein–Microtubule Network Is Required for NGF Signaling Through the Rap1/MAPK Pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen‐activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule‐ and dynein‐based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF‐induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein‐ and microtubule‐based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.

BPAG1n4 is essential for retrograde axonal transport in sensory neurons

Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice. We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo. Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4. Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain. Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein. Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport. We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons. These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.

Lysosome Biogenesis Mediated by vps-18 Affects Apoptotic Cell Degradation in Caenorhabditis elegans

Appropriate clearance of apoptotic cells (cell corpses) is an important step of programmed cell death. Although genetic and biochemical studies have identified several genes that regulate the engulfment of cell corpses, how these are degraded after being internalized in engulfing cell remains elusive. Here, we show that VPS-18, the Caenorhabditis elegans homologue of yeast Vps18p, is critical to cell corpse degradation. VPS-18 is expressed and functions in engulfing cells. Deletion of vps-18 leads to significant accumulation of cell corpses that are not degraded properly. Furthermore, vps-18 mutation causes strong defects in the biogenesis of endosomes and lysosomes, thus affecting endosomal/lysosomal protein degradation. Importantly, we demonstrate that phagosomes containing internalized cell corpses are unable to fuse with lysosomes in vps-18 mutants. Our findings thus provide direct evidence for the important role of endosomal/lysosomal degradation in proper clearance of apoptotic cells during programmed cell death.

PtdIns(4)P regulates retromer–motor interaction to facilitate dynein–cargo dissociation at the trans-Golgi network

The molecular mechanisms for the retrograde motor dynein–dynactin to unload its cargoes at their final destination remain to be elucidated. In this study, we have investigated the regulatory mechanism underlying release of retromer-associated cargoes at the trans-Golgi network (TGN). We report that phosphotidylinositol-4-phosphate (PtdIns(4)P), a Golgi-enriched phosphoinositide, negatively regulates the protein–protein interaction between the p150Glued subunit of dynein–dynactin and the retromer component SNX6. We show that PtdIns(4)P specifically facilitates dissociation of retromer-mediated membranous cargoes from the motor at the TGN and uncover an important function for PtdIns(4)P in the spatial control of retrograde vesicular trafficking to the TGN membrane. PtdIns(4)P also regulates SNX4-mediated retrograde vesicular trafficking to the endocytic recycling compartment by modulating its interaction with dynein. These results establish organelle-specific phosphoinositide regulation of motor–cargo interaction as a mechanism for cargo release by molecular motors at target membrane.

Retrolinkin, a membrane protein, plays an important role in retrograde axonal transport

Retrograde axonal transport plays an important role in the maintenance of neuronal functions, but the mechanism is poorly defined partly because the constituents of the retrograde transport system and their interactions have yet to be elucidated. Of special interest is how dynein/dynactin motor proteins interact with membrane cargoes. Here, we report that an endosomal vesicle protein, termed retrolinkin, functions as a receptor tethering vesicles to dynein/dynactin through BPAG1n4. Retrolinkin, a membrane protein highly enriched in neuronal endosomes, binds directly to BPAG1n4. Deletion of retrolinkin membrane-association domains disrupts retrograde vesicular transport, recapitulating the BPAG1 null phenotype. We propose that retrolinkin acts with BPAG1n4 to specifically regulate retrograde axonal transport. Our work lays the foundation for understanding fundamental issues of axonal transport and provides insights into the molecular mechanisms underlying human neurodegenerative disorders.