Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.     

Down-Regulation of a Manganese Transporter in the Face of Metal Toxicity

The yeast Smf1p Nramp manganese transporter is posttranslationally regulated by environmental manganese. Smf1p is stabilized at the cell surface with manganese starvation, but is largely degraded in the vacuole with physiological manganese through a mechanism involving the Rsp5p adaptor complex Bsd2p/Tre1p/Tre2p. We now describe an additional level of Smf1p regulation that occurs with toxicity from manganese, but not other essential metals. This regulation is largely Smf1p-specific. As with physiological manganese, toxic manganese triggers vacuolar degradation of Smf1p by trafficking through the multivesicular body. However, regulation by toxic manganese does not involve Bsd2p/Tre1p/Tre2p. Toxic manganese triggers both endocytosis of cell surface Smf1p and vacuolar targeting of intracellular Smf1p through the exocytic pathway. Notably, the kinetics of vacuolar targeting for Smf1p are relatively slow with toxic manganese and require prolonged exposures to the metal. Down-regulation of Smf1p by toxic manganese does not require transport activity of Smf1p, whereas such transport activity is needed for Smf1p regulation by manganese starvation. Furthermore, the responses to manganese starvation and manganese toxicity involve separate cellular compartments. We provide evidence that manganese starvation is sensed within the lumen of the secretory pathway, whereas manganese toxicity is sensed within an extra-Golgi/cytosolic compartment of the cell.     

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.     

Mutational analysis of Saccharomyces cerevisiae Smf1p, a member of the Nramp family of metal transporters.

We have recently shown that a member of the Nramp family of metal transporters, Saccharomyces cerevisiae Smf1p, is tightly regulated at the level of protein stability and protein sorting. Under metal replete conditions, Smf1p is targeted to the vacuole for degradation in a manner dependent on the S. cerevisiaeBSD2 gene product, but under metal starvation conditions, Smf1p accumulates at the cell surface. Here, we have addressed whether Smf1p activity may be necessary for its regulation by metal ions and Bsd2p. Well conserved residues within transmembrane domain 4 and the transport signature sequence of Smf1p were mutagenized. We identified two mutants, G190A and G424A, which destroyed Smf1p activity as monitored by complementation of a smf1 mutation. Notably, these mutations also abolished control by metal ions and Bsd2p, suggesting that Smf1p metal transport function may be necessary for its regulation. Two additional mutants isolated (Q419A and E423A) exhibited wild-type complementation activity and were properly targeted for vacuolar degradation in a Bsd2-dependent manner. However, these mutants failed to re-distribute to the plasma membrane under conditions of metal starvation. A model is proposed herein describing the probable role of Smf1 protein conformation in directing its movement to the vacuole versus cell surface in response to changes in metal ion availability.     

Arrestin-Mediated Endocytosis of Yeast Plasma Membrane Transporters

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.     

A novel role for Bsd2 in the resistance of yeast to adriamycin

In a search for undiscovered mechanisms of resistance to adriamycin, we screened a genomic library derived from Saccharomyces cerevisiae for genes related to adriamycin resistance. To our surprise, we found that overexpression of BSD2 rendered yeast cells resistant to adriamycin. Downregulation of the metal transporters Smf1 and Smf2 is the only activity of Bsd2 reported to date, and Bsd2 deficiency increases intracellular levels of Smf1 and Smf2. SMF2-disrupted cells exhibited significantly greater resistance to adriamycin, whereas the resistance of SMF1-disrupted cells was only slightly improved. The sensitivity of the SMF1- and SMF2-disrupted yeast cell line overexpressing BSD2 was almost the same as that of the BSD2-overexpressing parental yeast cell. Thus the overexpression of BSD2 and the disruption of SMF1 and SMF2 might be involved in the same mechanism that confers resistance to adriamycin. Although both SMF1- and SMF2-disrupted cells were very sensitive to EGTA, overexpression of BSD2 had little or no effect on sensitivity to EGTA. However, a partial decrease in the intracellular level of FLAG-Smf2 was observed by overexpression of BSD2. Thus, the resistance to adriamycin acquired by overexpression of BSD2 might be partially explained by down-regulation of Smf2, but in addition to Smf2, other as of yet unidentified targets of Bsd2 must also be responsible for the resistance.     

Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1

Many plasma membrane proteins in yeast are ubiquitinated and endocytosed, but how they are recognized for modification has remained unknown. Here, we show that the manganese transporter Smf1 is endocytosed when cells are exposed to cadmium ions, that this endocytosis depends on Rsp5-dependent ubiquitination of specific lysines and that it also requires phosphorylation at nearby sites. This phosphorylation is, however, constitutive rather than stress-induced. Efficient ubiquitination requires Ecm21 or Csr2, two members of a family of arrestin-like yeast proteins that contain several PY motifs and bind to Rsp5. Ecm21 also binds to phosphorylated Smf1, providing a link between Rsp5 and its substrate. PY motif-containing arrestin-like proteins are found in many species, including humans, and might have a general role as ubiquitin ligase adaptors.     

The family of SMF metal ion transporters in yeast cells

Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis. The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles. In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes. Each null mutation exhibited sensitivity to metal ion chelators at different concentrations. The combination of null mutants DeltaSMF1 + DeltaSMF2 and the triple null mutant Delta3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively. Addition of 5 microm copper or 25 microm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent. The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein. Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Delta3SMF. Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes. The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3 and FET4 were inactivated. Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium. We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells.     

Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p

Eukaryotes express both copper/zinc (SOD1)- and manganese (SOD2)-requiring superoxide dismutase enzymes that guard against oxidative damage. Although SOD1 acquires its copper through a specific copper trafficking pathway, nothing is known regarding the intracellular manganese trafficking pathway for SOD2. We demonstrate here that in Saccharomyces cerevisiae cells delivery of manganese to SOD2 in the mitochondria requires the Nramp metal transporter, Smf2p. SOD2 activity is greatly diminished in smf2Delta mutants, even though the mature SOD2 polypeptide accumulates to normal levels in mitochondria. Treating smf2Delta cells with manganese supplements corrected the SOD2 defect, as did elevating intracellular manganese through mutations in PMR1. Hence, manganese appears to be inaccessible to mitochondrial SOD2 in smf2 mutants. Cells lacking SMF2 also exhibited defects in manganese-dependent steps in protein glycosylation and showed an overall decrease in steady-state levels of accumulated manganese. By comparison, mutations in the cell surface Nramp transporter, Smf1p, had very little impact on manganese accumulation and trafficking. Smf2p resides in intracellular vesicles and shows no evidence of plasma membrane localization, even in an end4 mutant blocked for endocytosis. We propose a model in which Smf2p-containing vesicles play a central role in manganese trafficking to the mitochondria and other cellular sites as well.     

The overlapping roles of manganese and Cu/Zn SOD in oxidative stress protection

In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.     

Manganese Redistribution by Calcium-stimulated Vesicle Trafficking Bypasses the Need for P-type ATPase Function

Regulation of intracellular ion homeostasis is essential for eukaryotic cell physiology. An example is provided by loss of ATP2C1 function, which leads to skin ulceration, improper keratinocyte adhesion, and cancer formation in Hailey-Hailey patients. The yeast ATP2C1 orthologue PMR1 codes for a Mn²⁺/Ca²⁺ transporter that is crucial for cis-Golgi manganese supply. Here, we present evidence that calcium overcomes the lack of Pmr1 through vesicle trafficking-stimulated manganese delivery and requires the endoplasmic reticulum Mn²⁺ transporter Spf1 and the late endosome/trans-Golgi Nramp metal transporter Smf2. Smf2 co-localizes with the putative Mn²⁺ transporter Atx2, and ATX2 overexpression counteracts the beneficial impact of calcium treatment. Our findings suggest that vesicle trafficking promotes organelle-specific ion interchange and cytoplasmic metal detoxification independent of calcineurin signaling or metal transporter re-localization. Our study identifies an alternative mode for cis-Golgi manganese supply in yeast and provides new perspectives for Hailey-Hailey disease treatment.     

Frataxin depletion in yeast triggers up-regulation of iron transport systems before affecting iron-sulfur enzyme activities

The primary function of frataxin, a mitochondrial protein involved in iron homeostasis, remains controversial. Using a yeast model of conditional expression of the frataxin homologue YFH1, we analyzed the primary effects of YFH1 depletion. The main conclusion unambiguously points to the up-regulation of iron transport systems as a primary effect of YFH1 down-regulation. We observed that inactivation of aconitase, an iron-sulfur enzyme, occurs long after the iron uptake system has been activated. Decreased aconitase activity should be considered part of a group of secondary events promoted by iron overloading, which includes decreased superoxide dismutase activity and increased protein carbonyl formation. Impaired manganese uptake, which contributes to superoxide dismutase deficiency, has also been observed in YFH1-deficient cells. This low manganese content can be attributed to the down-regulation of the metal ion transporter Smf2. Low Smf2 levels were not observed in AFT1/YFH1 double mutants, indicating that high iron levels could be responsible for the Smf2 decline. In summary, the results presented here indicate that decreased iron-sulfur enzyme activities in YFH1-deficient cells are the consequence of the oxidative stress conditions suffered by these cells.     

Yeast Mn2+ transporter, Smf1p, is regulated by ubiquitin-dependent vacuolar protein sorting

Conditional cdc1(Ts) mutants of S. cerevisiae arrest with a phenotype similar to that exhibited by Mn(2+)-depleted cells. Sequence similarity between Cdc1p and a class of Mn(2+)-dependent phosphoesterases, as well as the observation that conditional cdc1(Ts) growth can be ameliorated by Mn(2+) supplement, suggests that Cdc1p activity is sensitive to intracellular Mn(2+) levels. This article identifies several previously uncharacterized cdc1(Ts) suppressors as class E vps (vacuolar protein sorting) mutants and shows that these, as well as other vps mutants, accumulate high levels of intracellular Mn(2+). Yeast VPS genes play a role in delivery of membrane transporters to the vacuole for degradation, and we show that the vps mutants accumulate elevated levels of the high-affinity Mn(2+) transporter Smf1p. cdc1(Ts) conditional growth is also alleviated by mutations, including doa4 and ubc4, that compromise protein ubiquitination, and these ubiquitination defects are associated with Smf1p accumulation. Epistasis studies show that these suppressors require functional Smf1p to alleviate the cdc1(Ts) growth defect, whereas Smf1p is dispensable for cdc1(Ts) suppression by a mutation (cos16/per1) that does not influence intracellular Mn(2+) levels. Because Smf1p is ubiquitinated in vivo, we propose that Smf1p is targeted to the vacuole for degradation by ubiquitination-dependent protein sorting.     

A 'distributed degron' allows regulated entry into the ER degradation pathway.

Protein degradation is employed in both regulation and quality control. Regulated degradation of specific proteins is often mediated by discrete regions of primary sequence known as degrons, whereas protein quality control involves recognition of structural features common to damaged or misfolded proteins, rather than specific features of an individual protein. The yeast HMG-CoA reductase isozyme Hmg2p undergoes stringently regulated degradation by machinery that is also required for ER quality control. The 523 residue N-terminal transmembrane domain of Hmg2p is necessary and sufficient for regulated degradation. To understand how Hmg2p undergoes regulated degradation by the ER quality control pathway, we analyzed over 300 mutants of Hmg2p. Regulated degradation of Hmg2p requires information distributed over the entire transmembrane domain. Accordingly, we refer to this determinant as a 'distributed' degron, which has functional aspects consistent with both regulation and quality control. The Hmg2p degron functions in the specific, regulated degradation of Hmg2p and can impart regulated degradation to fusion proteins. However, its recognition is based on dispersed structural features rather than primary sequence motifs. This mode of targeting has important consequences both for the prediction of degradation substrates and as a potential therapeutic strategy for targeted protein degradation using endogenous degradation pathways.     

Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast

Cystic fibrosis is the most widespread hereditary disease among the white population caused by different mutations of the apical membrane ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR). Its most common mutation, DeltaF508, leads to nearly complete degradation via endoplasmic reticulum-associated degradation (ERAD). Elucidation of the quality control and degradation mechanisms might give rise to new therapeutic approaches to cure this disease. In the yeast Saccharomyces cerevisiae, a variety of components of the protein quality control and degradation system have been identified. Nearly all of these components share homology with mammalian counterparts. We therefore used yeast mutants defective in the ERAD system to identify new components that are involved in human CFTR quality control and degradation. We show the role of the lectin Htm1p in the degradation process of CFTR. Complementation of the HTM1 deficiency in yeast cells by the mammalian orthologue EDEM underlines the necessity of this lectin for CFTR degradation and highlights the similarity of quality control and ERAD in yeast and mammals. Furthermore, degradation of CFTR requires the ubiquitin protein ligases Der3p/Hrd1p and Doa10p as well as the cytosolic trimeric Cdc48p-Ufd1p-Npl4p complex. These proteins also were found to be necessary for ERAD of a mutated yeast "relative" of CFTR, Pdr5(*)p.     

Interactions between AMOT PPxY motifs and NEDD4L WW domains function in HIV-1 release

Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1–NEDD4L WW3 interaction accounts for most of the AMOT–NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW–PPxY core interaction account for the unusually high affinity of the AMOT PPxY1–NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.     

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.     

Recycling endosomes contribute to autophagosome formation

Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources.