A receptor domain controls the intracellular sorting of the ferrichrome transporter, ARN1

The Saccharomyces cerevisiae transporter Arn1p takes up the ferric-siderophore ferrichrome, and extracellular ferrichrome dramatically influences the intracellular trafficking of Arn1p. In the absence of ferrichrome, Arn1p sorts directly to the endosomal compartment. At low concentrations of ferrichrome, Arn1p stably relocalizes to the plasma membrane, yet little to no uptake of ferrichrome occurs at these low concentrations. At higher concentrations of ferrichrome, Arn1p cycles between the plasma membrane and endosome. Arn1p contains two binding sites for ferrichrome: one site has an affinity similar to the K(T) for transport, but the second site has a much higher affinity. Here we report that this high-affinity binding site lies within a unique extracytosolic, carboxyl-terminal domain. Mutations within this domain lead to loss of ferrichrome binding and uptake activities and missorting of Arn1p, including a failure to relocalize to the plasma membrane in the presence of ferrichrome. Mutation of phenylalanine residues in the cytosolic tail of Arn1p also lead to missorting, but without defects in ferrichrome binding. We propose that the carboxyl terminus of Arn1p contains a receptor domain that controls the intracellular trafficking of the transporter.     

Ferrichrome induces endosome to plasma membrane cycling of the ferrichrome transporter,Arn1p,in Saccharomyces cerevisiae

Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore-iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of ferrichrome-iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.     

GGA2- and ubiquitin-dependent trafficking of Arn1, the ferrichrome transporter of Saccharomyces cerevisiae

The intracellular trafficking of Arn1, a ferrichrome transporter in Saccharomyces cerevisiae, is controlled in part by the binding of ferrichrome to the transporter. In the absence of ferrichrome, Arn1 is sorted directly from the Golgi to endosomes. Ferrichrome binding triggers the redistribution of Arn1 to the plasma membrane, whereas ferrichrome transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. Here, we report that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi-to-endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, but ubiquitination was not required for plasma membrane accumulation of Arn1 in the presence of ferrichrome. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. High concentrations of ferrichrome led to higher levels of ubiquitination of Arn1, but they did not induce degradation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with polyubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter.     

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.     

Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiae

Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified. These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate. Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media. However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed. Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p. Moreover the available nitrogen source regulates the expression of SER genes. However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2. Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose. Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.     

SorLA/LR11 regulates processing of amyloid precursor protein via interaction with adaptors GGA and PACS-1

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.     

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBD-labeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50Delta background. Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.     

Kin1 is a plasma membrane‐associated kinase that regulates the cell surface in fission yeast

Cell morphogenesis is a complex process that depends on cytoskeleton and membrane organization, intracellular signalling and vesicular trafficking. The rod shape of the fission yeast Schizosaccharomyces pombe and the availability of powerful genetic tools make this species an excellent model to study cell morphology. Here we have investigated the function of the conserved Kin1 kinase. Kin1‐GFP associates dynamically with the plasma membrane at sites of active cell surface remodelling and is present in the membrane fraction. Kin1Δ null cells show severe defects in cell wall structure and are unable to maintain a rod shape. To explore Kin1 primary function, we constructed an ATP analogue‐sensitive allele kin1‐as1. Kin1 inhibition primarily promotes delocalization of plasma membrane‐associated markers of actively growing cell surface regions. Kin1 itself is depolarized and its mobility is strongly reduced. Subsequently, amorphous cell wall material accumulates at the cell surface, a phenotype that is dependent on vesicular trafficking, and the cell wall integrity mitogen‐activated protein kinase pathway is activated. Deletion of cell wall integrity mitogen‐activated protein kinase components reduces kin1Δ hypersensitivity to stresses such as those induced by Calcofluor white and SDS. We propose that Kin1 is required for a tight link between the plasma membrane and the cell wall.     

Visualizing differences in ligand-induced beta-arrestin-GFP interactions and trafficking between three recently characterized G protein-coupled receptors

beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.     

Non-SCF-type F-box protein Roy1/Ymr258c interacts with a Rab5-like GTPase Ypt52 and inhibits Ypt52 function

Skp1/Cul1/F-box (SCF)-type F-box proteins are a component of the Cullin-RING SCF ubiquitin E3 ligase, which is involved in numerous cellular processes. However, the function of non-SCF-type F-box proteins remains largely unknown. The Rab5-like small guanosine 5'-triphosphatase Vps21/Ypt51 is a key regulator of intracellular transportation; however, deletion of its isoforms, Ypt52 and Ypt53, results in only a modest inhibition of intracellular trafficking. The function of these proteins therefore remains largely elusive. Here we analyze the role of a previously uncharacterized non-SCF-type F-box protein, Roy1/Ymr258c, in cell growth and intracellular transport in Saccharomyces cerevisiae. Roy1 binds to Ypt52 under physiological conditions, and Skp1 is indispensable for the association of Roy1 with Ypt52. The vps21Δ yeast cells exhibit severe deficiencies in cell growth and intracellular trafficking, whereas simultaneous deletion of roy1 alleviates the defects caused by deletion of vps21. However, additional disruption of ypt52 in roy1Δvps21Δ cells largely suppresses the cell growth and trafficking observed in roy1Δvps21Δ cells. We demonstrate that Roy1 interacts with guanosine 5'-diphosphate-bound and nucleotide-free Ypt52 and thereby inhibits the formation of guanosine 5'-triphosphate-bound, active Ypt52. These results thus indicate that Roy1 negatively modulates cell viability and intracellular transport by suppressing Ypt52.     

CUE domain-containing protein Vps901 is required for vacuolar protein transport in Schizosaccharomyces pombe

The functions of two Schizosaccharomyces pombe Vps9-like genes, SPBC4F6.10/vps901(+) and SPBC29A10.11c/vps902(+), were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901(+) resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902(+) had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901(+) further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Internalization and trafficking of fluorescent-labeled phospholipids in yeast

Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored. This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes. This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach.     

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.     

Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein in the hepatocyte. It is expressed as two spliced isoforms differing by the presence (full length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Because the two isoforms differ by their ability to regulate receptor-mediated insulin endocytosis and degradation, we aimed to investigate the cellular basis for this functional difference by comparing their intracellular trafficking. During its intracellular assembly, pp120 is transported from the trans-Golgi network to the sinusoidal domain of the plasma membrane before its final transcytosis to the bile canalicular domain. Because both isoforms are expressed in hepatocytes, we examined their intracellular trafficking in NIH 3T3 fibroblasts individually transfected with each isoform. Pulse-chase experiments demonstrated that most of the newly synthesized full-length isoform reached complete maturation at about 60 min of chase. By contrast, only about 40% of the newly synthesized truncated isoform underwent complete maturation, even at more prolonged chase. Moreover, a significant portion of the truncated isoform appeared to be targeted to lysosomes. Abolishing basal phosphorylation on Ser(503) by cAMP-dependent serine kinase by mutating this residue to alanine was correlated with incomplete maturation of full length pp120 in NIH 3T3 cells and hepatocytes. This finding suggests that the intracellular domain of pp120 contains information that regulates its vectorial sorting from the trans-Golgi network to the plasma membrane.     

Role for two conserved intermembrane space proteins, Ups1p and Up2p, in intra-mitochondrial phospholipid trafficking

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.     

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.