Subcellular distribution of human tyrosine hydroxylase isoforms 1 and 4 in SH-SY5Y cells

Tyrosine hydroxylase (TH) is the key enzyme that controls the rate of synthesis of the catecholamines. SH-SY5Y cells with stable transfections of either human tyrosine hydroxylase isoform 1 (hTH1) or human tyrosine hydroxylase isoform 4 (hTH4) were used to determined the subcellular distribution of TH protein and phosphorylated TH, under basal conditions and after muscarine stimulation. Muscarine was previously shown to increase the phosphorylation of only serine 19 and serine 40 in hTH1 cells. Under basal conditions, the hTH1 and hTH4 proteins, their serine 19 phosphorylated forms and hTH1 phosphorylated at serine 40 were all similarly distributed; with ~80% in the cytosolic fraction, ~20% in the membrane fraction, and less than 1%, or not detectable, in the nuclear fraction. However, hTH4 phosphorylated at serine 71 had a significantly different distribution with ~65% cytosolic and ~35% membrane associated. Muscarine stimulation led to hTH1 being redistributed from the cytosol and nuclear fractions to the membrane fraction and hTH4 being redistributed from the cytosol to the nuclear fraction. These muscarine stimulated redistributions were not due to TH phosphorylation at serine 19, serine 40, or serine 71 and were most likely due to TH binding to proteins whose phosphorylation was increased by muscarine. This is the first study to show a difference in subcellular distribution between two human TH isoforms under basal and stimulated conditions.     

Direct proteasome inhibition by clasto-lactacystin beta-lactone permits the detection of ubiquitinated p21(waf1) in ML-1 cells.

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.     

Proteasome inhibition induces TNFR1 shedding from human airway epithelial (NCI-H292) cells

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.     

Sna3 Is an Rsp5 Adaptor Protein that Relies on Ubiquitination for Its MVB Sorting

The process in which ubiquitin ( Ub ) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies ( MVBs ) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However , Ub ‐independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna 3, the prototypical Ub ‐independent cargo protein sorted through the MVB pathway in yeast. We show that Sna 3 trafficking to the vacuole is critically dependent on Rsp 5 ligase activity and ubiquitination. We find Sna 3 undergoes Ub ‐dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna 3 as an adaptor protein that recruits Rsp 5 to cargo such as the methionine transporter Mup 1, resulting in efficient Mup 1 delivery to the vacuole .     

Food Vacuole Associated Enolase in Plasmodium Undergoes Multiple Post-Translational Modifications: Evidence for Atypical Ubiquitination

Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH₂-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P . yoelii . The composition of food vacuolar proteome and likely interactors of enolase are also being reported.     

The mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells

The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38; (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38; (iii) S40D, Ser40 replaced by Asp40; and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE > S40D > S40A = RR-GG > wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.     

DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.     

Evidence for a functionally important histidine residue in human tyrosine hydroxylase

Summary Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M^–1 min^–1 at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per enzyme subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoenzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.Abbreviations BH₄ (6R)-l-erythro-tetrahydrobiopterin - DEP diethylpyrocarbonate - DOPA 3,4-dihydroxyphenylalanine - hTH1 human tyrosine hydroxylase isoenzyme 1 - apo-hTH1 apoenzyme of hTH1 - Fe(II)-hTH1 holoenzyme (iron reconstituted) of hTH1 - dopamine-Fe(III)-hTH1 holoenzyme of hTH1 with dopamine bound - TH tyrosine hydroxylase     

Spatiotemporal Regulation of the Ubiquitinated Cargo-binding Activity of Rabex-5 in the Endocytic Pathway

Ubiquitin (Ub)-dependent endocytosis of membrane proteins requires precise molecular recognition of ubiquitinated cargo by Ub-binding proteins (UBPs). Many UBPs are often themselves monoubiquitinated, a mechanism referred to as coupled monoubiquitination, which prevents them from binding in trans to the ubiquitinated cargo. However, the spatiotemporal regulatory mechanism underlying the interaction of UBPs with the ubiquitinated cargo, via their Ub-binding domains (UBDs) remains unclear. Previously, we reported the interaction of Rabex-5, a UBP and guanine nucleotide exchange factor (GEF) for Rab5, with ubiquitinated neural cell adhesion molecule L1, via its motif interacting with Ub (MIU) domain. This interaction is critical for the internalization and sorting of the ubiquitinated L1 into endosomal/lysosomal compartments. The present study demonstrated that the interaction of Rabex-5 with Rab5 depends specifically on interaction of the MIU domain with the ubiquitinated L1 to drive its internalization. Notably, impaired GEF mutants and the Rabex-5^E213A mutant increased the flexibility of the hinge region in the HB-VPS9 tandem domain, which significantly affected their interactions with the ubiquitinated L1. In addition, GEF mutants increased the catalytic efficiency, which resulted in a reduced interaction with the ubiquitinated L1. Furthermore, the coupled monoubiquitination status of Rabex-5 was found to be significantly associated with interaction of Rabex-5 and the ubiquitinated L1. Collectively, our study reveals a novel mechanism, wherein the GEF activity of Rabex-5 acts as an intramolecular switch orchestrating ubiquitinated cargo-binding activity and coupled monoubiquitination to permit the spatiotemporal dynamic exchange of the ubiquitinated cargos.     

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.     

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.     

Recognition of modified forms of ribonuclease A by the ubiquitin system

The substrate specificity of the ubiquitin (Ub) conjugation system was explored with regard to recognition of unfolded conformation and/or oxidized methionine residues in six derivatives of bovine RNase A. Based on the following observations, ubiquitination of RNase A substrates by the enzymes in a rabbit reticulocyte extract appears to correlate with unfolded conformation rather than with methionine oxidation. 1) Methionine oxidation in already unfolded forms of RNase A does not enhance ubiquitination. 2) Fluorescence measurements and iodoacetate trapping of free sulfhydryls show that the disulfide bonds of MetSO-RNase A, in which the 4 methionine residues are oxidized to the sulfoxide, are reduced by 2 mM dithiothreitol (DTT) in standard Ub conjugation assays so that this derivative also is unfolded. 3) Although MetSO-RNase A is ubiquitinated in the absence of DTT, its intrinsic fluorescence, cation-exchange properties, and susceptibility to reduction indicate a non-native conformation. 4) Methionine sulfoxide-containing peptides that mimic regions of RNase A fail to inhibit conjugation of 125I-Ub to MetSO-RNase A. Ub adducts to two of the six derivatives (MetSO- and reduced/carboxamidomethylated MetSO-RNase A) increase when DTT is omitted from the reactions. Ubaldehyde, an inhibitor of isopeptidases that disassemble Ub-protein conjugates, increased product yields and reduced or abolished the DTT effect, suggesting that an isopeptidase specific for these two RNase A derivatives may be inactivated by oxidation. Ub conjugates of the other RNase A derivatives also increase with Ub-aldehyde but are unaffected by DTT.     

Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins

The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.     

The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs

Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery. Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA). GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein. Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes. Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin. These motifs are found within different helices within the three-helix GAT subdomain. When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.     

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.