Rainer Duden

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.

β-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the golgi complex, shows homology to β-adaptin

Abstract We have cloned and sequenced β-COP, a peripheral 110 kd Golgi membrane protein. β-COP shows significant homology to β-adaptin. It is present in a membrane-bound form and in a cytosolic complex of 13–14S, with a Stokes radius of ∼10 nm and an estimated M r of ∼550,000. By immunofluorescence labeling, β-COP is associated with the structures of the Golgi complex. Immunoelectron microscopy has localized β-COP to non-clathrin-coated vesicles and cisternae of the Golgi complex. These coated vesicles accumulate in rat liver Golgi fractions treated with GTPγS and strongly label for-COP Our data suggest that β-COP is a component of a coat associated with vesicles and cisternae of the Golgi complex.

The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins.

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.

COP I domains required for coatomer integrity, and novel interactions with ARF and ARF‐GAP

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two‐hybrid system, we characterize the domain structure of the α‐, β′‐, ϵ‐COP and β‐, γ‐, δ‐, ζ‐COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the β‐, γ‐, δ‐, ζ‐COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of α‐COP truncation mutants, we characterize distinct functional domains on α‐COP. Its N‐terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX‐dependent trafficking. The last ∼170 amino acids of α‐COP are also non‐essential for cell viability, but required for ϵ‐COP incorporation into coatomer and maintainance of normal ϵ‐COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: β′‐ and γ‐COP interact with the ARF‐GTP‐activating protein (GAP) Glo3p, but not Gcs1p, and β‐ and ϵ‐COP interact with ARF‐GTP. Glo3p also interacts with intact coatomer in vitro.

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine‐tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2–1 and ret3–1 were defective for ER retrieval, only ret2–1 exhibited a defect in forward ER‐to‐Golgi transport at the non‐permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine‐tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.

ER-to-Golgi transport: COP I and COP II function (Review)

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex.

ARF-GAP–mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)–GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.

epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.

We isolated a novel yeast α‐COP mutant, ret1‐3, in which α‐COP is degraded after cells are shifted to a restrictive temperature. ret1‐3 cells cease growth at 28°C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast ϵ‐COP gene. ϵ‐COP (Sec28p) overproduction suppresses the defects of ret1‐3 cells up to 34°C, through stabilizing levels of α‐COP. Surprisingly, cells lacking ϵ‐COP (sec28Δ) grow well up to 34°C and display normal trafficking of carboxypeptidase Y and KKXX‐tagged proteins at a permissive temperature. ϵ‐COP is thus non‐essential for yeast cell growth, but sec28Δ cells are thermosensitive. In sec28Δ cells shifted to 37°C, wild‐type α‐COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by α‐COP overproduction. Mutant coatomer from sec28Δ cells behaves as an unusually large protein complex in gel filtration experiments. The sec28Δ mutation displays allele‐specific synthetic‐lethal interactions with α‐COP mutations: sec28Δ ret1‐3 double mutants are unviable at all temperatures, whereas sec28Δ ret1‐1 double mutants grow well up to 30°C. Our results suggest that a function of ϵ‐COP is to stabilize α‐COP and the coatomer complex.

Gamma-COP appendage domain - structure and function

COPI‐coated vesicles mediate retrograde transport from the Golgi back to the ER and intra‐Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β‐, γ‐, δ‐ and ζ‐COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α‐, β′‐ and ε‐COP subunits. Here, we present the structure of the appendage domain of γ‐COP and show that it has a similar overall fold as the α‐appendage of AP2. Again, like the α‐appendage the γ‐COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ‐COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ‐COP, Sec21p, a second binding site is proposed to exist on the γ‐COP appendage that interacts with the α,β′,ε COPI subcomplex.

Two Human ARFGAPs Associated with COP‐I‐Coated Vesicles

ADP‐ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP‐ribosylation factor‐GTPase‐activating proteins (ARFGAPs) are proposed to contribute to site‐specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)‐I‐dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP‐I‐coated vesicles generated from Golgi membranes in the presence of GTP‐γ‐S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (ΔN‐ARFGAP2) inhibits COP‐I‐dependent Golgi‐to‐endoplasmic reticulum transport of cholera toxin (CTX‐K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.