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1.  ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies 
Journal of Experimental Botany  2013;64(10):2817-2829.
Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1–2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP–ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP–ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP–ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.
doi:10.1093/jxb/ert125
PMCID: PMC3697957  PMID: 23682115
ARA7(Q69L); homotypic fusion; MVB enlargement; multivesicular body; pre-vacuolar compartment; transgenic Arabidopsis cells.
2.  New insights into the role of Arabidopsis RABA1 GTPases in salinity stress tolerance 
Plant Signaling & Behavior  2013;8(9):e25377.
RAB11 GTPases, widely conserved members of RAB small GTPases, have evolved in a unique way in plants; plant RAB11 has notable diversity compared with animals and yeast. Recently, we have shown that members of RABA1, a subgroup in Arabidopsis RAB11 group, are required for salinity stress tolerance. To obtain a clue to understand its underlying mechanism, here we investigate whether RABA1 regulates sodium transport across the plasma membrane and accumulation in the vacuole. The results indicate that the raba1 quadruple mutant is not defective in the import and intracellular distribution of sodium, implying that RABA1 members are involved in a more indirect way in the responses to salinity stress.
doi:10.4161/psb.25377
PMCID: PMC4002589  PMID: 23803751
RAB GTPase; RABA1; salinity stress
3.  A Unique HEAT Repeat-Containing Protein SHOOT GRAVITROPISM6 is Involved in Vacuolar Membrane Dynamics in Gravity-Sensing Cells of Arabidopsis Inflorescence Stem 
Plant and Cell Physiology  2014;55(4):811-822.
Plant vacuoles play critical roles in development, growth and stress responses. In mature cells, vacuolar membranes (VMs) display several types of structures, which are formed by invagination and folding of VMs into the lumenal side and can gradually move and change shape. Although such VM structures are observed in a broad range of tissue types and plant species, the molecular mechanism underlying their formation and maintenance remains unclear. Here, we report that a novel HEAT-repeat protein, SHOOT GRAVITROPISM6 (SGR6), of Arabidopsis is involved in the control of morphological changes and dynamics of VM structures in endodermal cells, which are the gravity-sensing cells in shoots. SGR6 is a membrane-associated protein that is mainly localized to the VM in stem endodermal cells. The sgr6 mutant stem exhibits a reduced gravitropic response. Higher plants utilize amyloplast sedimentation as a means to sense gravity direction. Amyloplasts are surrounded by VMs in Arabidopsis endodermal cells, and the flexible and dynamic structure of VMs is important for amyloplast sedimentation. We demonstrated that such dynamic features of VMs are gradually lost in sgr6 endodermal cells during a 30 min observation period. Histological analysis revealed that amyloplast sedimentation was impaired in sgr6. Detailed live-cell imaging analyses revealed that the VM structures in sgr6 had severe defects in morphological changes and dynamics. Our results suggest that SGR6 is a novel protein involved in the formation and/or maintenance of invaginated VM structures in gravity-sensing cells.
doi:10.1093/pcp/pcu020
PMCID: PMC3982123  PMID: 24486761
Arabidopsis; Gravitropism; HEAT-repeat protein; Vacuolar membrane
4.  Visualizing structural dynamics of thylakoid membranes 
Scientific Reports  2014;4:3768.
To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of photosystems I and II. However, direct evidence for such protein reorganization has not been visualized in live cells. Here we demonstrate structural dynamics of thylakoid membranes by live cell imaging in combination with deconvolution. We observed chlorophyll fluorescence in the antibiotics-induced macrochloroplast in the moss Physcomitrella patens. The three-dimensional reconstruction uncovered the fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in vivo. Our observation indicates that grana serve as a framework to maintain structural integrity of the entire thylakoid membrane network. Both the structural stability and flexibility of thylakoid membranes would be essential for dynamic protein reorganization under fluctuating light environments.
doi:10.1038/srep03768
PMCID: PMC3895878  PMID: 24442007
5.  Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy 
Scientific Reports  2013;3:2833.
Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes.
doi:10.1038/srep02833
PMCID: PMC3789154  PMID: 24088948
6.  The physiological role of SYP4 in the salinity and osmotic stress tolerances 
Plant Signaling & Behavior  2012;7(9):1118-1120.
The trans-Golgi network (TGN) contains multiple sorting domains and acts as the compartment for cargo sorting. Recent evidence indicates that the TGN also functions as an early endosome, the first compartment in the endocytic pathway in plants. The SYP4 group, plant Qa-SNAREs localized on the TGN, regulates both secretory and vacuolar transport pathways. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance to fungal pathogens. However, the physiological role of SYP4 in abiotic stress remains unknown. Here, we report the phenotypes of a syp4-mutant in regard to salinity and osmotic response, and describe the physiological roles of the SYP4 group in the abiotic stress response.
doi:10.4161/psb.21307
PMCID: PMC3489641  PMID: 22899062
SNARE; SYP4; TGN; salinity stress
7.  cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells 
Molecular Biology of the Cell  2012;23(16):3203-3214.
Particular cis-Golgi proteins accumulate in novel punctate structures close to ERES by BFA treatment in tobacco BY-2 cells. These structures reassemble first to form cis-Golgi after BFA removal, and the Golgi stacks regenerate in the cis-to-trans order. This indicates that the punctate structures act as the scaffold for Golgi regeneration.
The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.
doi:10.1091/mbc.E12-01-0034
PMCID: PMC3418314  PMID: 22740633
8.  Flowering Time Modulation by a Vacuolar SNARE via FLOWERING LOCUS C in Arabidopsis thaliana 
PLoS ONE  2012;7(7):e42239.
The transition of plant growth from vegetative to reproductive phases is one of the most important and dramatic events during the plant life cycle. In Arabidopsis thaliana, flowering promotion involves at least four genetically defined regulatory pathways, including the photoperiod-dependent, vernalization-dependent, gibberellin-dependent, and autonomous promotion pathways. Among these regulatory pathways, the vernalization-dependent and autonomous pathways are integrated by the expression of FLOWERING LOCUS C (FLC), a negative regulator of flowering; however, the upstream regulation of this locus has not been fully understood. The SYP22 gene encodes a vacuolar SNARE protein that acts in vacuolar and endocytic trafficking pathways. Loss of SYP22 function was reported to lead to late flowering in A. thaliana plants, but the mechanism has remained completely unknown. In this study, we demonstrated that the late flowering phenotype of syp22 was due to elevated expression of FLC caused by impairment of the autonomous pathway. In addition, we investigated the DOC1/BIG pathway, which is also suggested to regulate vacuolar/endosomal trafficking. We found that elevated levels of FLC transcripts accumulated in the doc1-1 mutant, and that syp22 phenotypes were exaggerated with a double syp22 doc1-1 mutation. We further demonstrated that the elevated expression of FLC was suppressed by ara6-1, a mutation in the gene encoding plant-unique Rab GTPase involved in endosomal trafficking. Our results indicated that vacuolar and/or endocytic trafficking is involved in the FLC regulation of flowering time in A. thaliana.
doi:10.1371/journal.pone.0042239
PMCID: PMC3407077  PMID: 22848750
9.  Qualitative difference between “bulb” membranes and other vacuolar membranes 
Plant Signaling & Behavior  2011;6(12):1914-1917.
“Bulb” is a mobile and complex structure appearing in vacuolar membrane of plant cell. We recently reported new fluorescent marker lines for bulbs and bulb-less mutants. We tried multicolor visualization of vacuolar membrane to show distinct segregation of bulb-positive protein (γTIP or AtVAM3) and bulb-negative protein (AtRab75). Unexpectedly, GFP-AtRab75 resulted to localize in bulb under the condition of co-expression with TagRFP-AtVAM3. The signal intensities of GFP-AtRab75 and TagRFP-AtVAM3 were quantified and compared. The result indicates that TagRFP-AtVAM3 is concentrated in bulb than GFP-AtRab75.
doi:10.4161/psb.6.12.18061
PMCID: PMC3337177  PMID: 22105033
AtRab75; AtVam3; plant growth; Rab-GTPase; SNARE; vacuolar membranes; “bulb”
10.  Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants 
Small GTPases  2012;3(1):23-27.
Lineage-specific expansion, followed by functional diversification of key components that act in membrane trafficking, is thought to contribute to lineage-specific diversification of organelles and membrane trafficking pathways. Indeed, recent comparative genomic studies have indicated that specific expansion of RAB and SNARE molecules occurred independently in various eukaryotic lineages over evolutionary history. However, experimental verification of this notion is difficult, because detailed functional analyses of RAB and SNARE proteins uniquely acquired by specific lineages are essential to understanding how new membrane trafficking pathways may have evolved. Recently, we found that a plant-specific RAB GTPase, ARA6, and a plant-unique R-SNARE, VAMP727, mediate a trafficking pathway from endosomes to the plasma membrane in Arabidopsis thaliana. Although a similar endosomal trafficking pathway was also reported in animals, the molecular machineries acting in these trafficking systems differ between animals and plants. Thus, trafficking pathways from endosomes to the plasma membrane appear to have been acquired independently in animal and plant systems. We further demonstrated that the ARA6-mediated trafficking pathway is required for the proper salt-stress response of A. thaliana. These results indicate that acquisition of a new membrane trafficking pathway may be associated with maximization of the fitness of each organism in a lineage-specific manner.
doi:10.4161/sgtp.18299
PMCID: PMC3398913  PMID: 22710734
ARA6; Arabidopsis thaliana; Rab5; SNARE; endosome; stress response
11.  Plant-Specific Myosin XI, a Molecular Perspective 
In eukaryotic cells, organelle movement, positioning, and communications are critical for maintaining cellular functions and are highly regulated by intracellular trafficking. Directional movement of motor proteins along the cytoskeleton is one of the key regulators of such trafficking. Most plants have developed a unique actin–myosin system for intracellular trafficking. Although the composition of myosin motors in angiosperms is limited to plant-specific myosin classes VIII and XI, there are large families of myosins, especially in class XI, suggesting functional diversification among class XI members. However, the molecular properties and regulation of each myosin XI member remains unclear. To achieve a better understanding of the plant-specific actin–myosin system, the characterization of myosin XI members at the molecular level is essential. In the first half of this review, we summarize the molecular properties of tobacco 175-kDa myosin XI, and in the later half, we focus on myosin XI members in Arabidopsis thaliana. Through detailed comparison of the functional domains of these myosins with the functional domain of myosin V, we look for possible diversification in enzymatic and mechanical properties among myosin XI members concomitant with their regulation.
doi:10.3389/fpls.2012.00211
PMCID: PMC3437519  PMID: 22973289
myosin XI; cytoplasmic streaming; intracellular transport; plants
12.  Membrane Traffic Within the Golgi Apparatus 
Newly synthesized secretory cargo molecules pass through the Golgi apparatus while resident Golgi proteins remain in the organelle. However, the pathways of membrane traffic within the Golgi are still uncertain. Most of the available data can be accommodated by the cisternal maturation model, which postulates that Golgi cisternae form de novo, carry the secretory cargoes forward, and ultimately disappear. The entry face of the Golgi receives material that has been exported from transitional ER sites, and the exit face of the Golgi is intimately connected with endocytic compartments. These conserved features are enhanced by cell type-specific elaborations such as tubular connections between mammalian Golgi cisternae. Key questions remain about how Golgi cisternae form and then mature, how resident Golgi proteins recycle, how Golgi compartments achieve their identities, how the unique architecture of the Golgi is established, and how different structural elements contribute to Golgi function.
doi:10.1146/annurev.cellbio.24.110707.175421
PMCID: PMC2877624  PMID: 19575639
cisternal maturation; COPI; transitional ER; recycling endosomes; Golgi matrix; Golgi tubules
13.  Journeys through the Golgi—taking stock in a new era 
The Journal of Cell Biology  2009;187(4):449-453.
The Golgi apparatus is essential for protein sorting and transport. Many researchers have long been fascinated with the form and function of this organelle. Yet, despite decades of scrutiny, the mechanisms by which proteins are transported across the Golgi remain controversial. At a recent meeting, many prominent Golgi researchers assembled to critically evaluate the core issues in the field. This report presents the outcome of their discussions and highlights the key open questions that will help guide the field into a new era.
doi:10.1083/jcb.200909011
PMCID: PMC2779233  PMID: 19948493
14.  Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions 
Nature  2008;456(7224):962-966.
Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication—a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms2–7. Polar PIN localization determines the direction of intercellular auxin flow8, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.
doi:10.1038/nature07409
PMCID: PMC2692841  PMID: 18953331
15.  Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha 
Plant and Cell Physiology  2009;50(6):1041-1048.
Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.
doi:10.1093/pcp/pcp055
PMCID: PMC2694730  PMID: 19369273
Actin; Arabidopsis thaliana; Lifeact; Liverwort; Marchantia polymorpha
16.  Phospholipid mediated plasticity in exocytosis observed in PC12 cells 
Brain research  2007;1151:46-54.
Membrane composition serves to identify intracellular compartments, signal cell death, as well as to alter a cell’s electrical and physical properties. Here we use amperometry to show that supplementation with the phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and phosphatidylserine (PS) can alter several aspects of exocytosis. Changes in the amperometric peak shape derived from individual exocytosing vesicles reveal that PC slows expulsion of neurotransmitter while PE accelerates expulsion of neurotransmitter. Amperometry data reveal a reduced amount of catecholamine released per event from PC-treated cells while electron micrographs indicate the vesicles in these cells are 50% larger than controls, thus providing evidence of pharmacological changes in vesicle concentration. Addition of SM appears to affect the rate of fusion pore expansion, indicated by slower peak rise times, but does not affect decay times or quantal size. Addition of PS results in a 1.7-fold increase in the number of events elicited by high-K+ depolarization. Electron micrographs of PS-treated cells suggest that increased vesicle recruitment underlies enhanced secretion. We did not observe any effect of phosphatidylinositol (PI) treatment. Together these data suggest that differences in membrane composition affect exocytosis and might be involved in mechanisms of cell function controlling the dynamics of communication via exocytosis.
doi:10.1016/j.brainres.2007.03.012
PMCID: PMC2034201  PMID: 17408597
Dopamine; Membrane phospholipid; Exocytosis; Plasticity; PC12 cell
17.  Smy2p Participates in COPII Vesicle Formation Through the Interaction with Sec23p/Sec24p Subcomplex 
Traffic (Copenhagen, Denmark)  2007;9(1):79-93.
The coat protein complex II (COPII) is essential for vesicle formation from the endoplasmic reticulum (ER) and is composed of two heterodimeric subcomplexes, Sec23p/Sec24p and Sec13p/Sec31p, and the small guanosine triphosphatase Sar1p. In an effort to identify novel factors that may participate in COPII vesicle formation, we isolated SMY2, a yeast gene encoding a protein of unknown function, as a multicopy suppressor of the temperature-sensitive sec24-20 mutant. We found that even a low-copy expression of SMY2 was sufficient for the suppression of the sec24-20 phenotypes, and the chromosomal deletion of SMY2 led to a severe growth defect in the sec24-20 background. In addition, SMY2 exhibited genetic interactions with several other genes involved in the ER-to-Golgi transport. Subcellular fractionation analysis showed that Smy2p was a peripheral membrane protein fractionating together with COPII components. However, Smy2p was not loaded onto COPII vesicles generated in vitro. Interestingly, coimmunoprecipitation between Smy2p and the Sec23p/Sec24p subcomplex was specifically observed in sec23-1 and sec24-20 backgrounds, suggesting that this interaction was a prerequisite for the suppression of the sec24-20 phenotypes by overexpression of SMY2. We propose that Smy2p is located on the surface of the ER and facilitates COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex.
doi:10.1111/j.1600-0854.2007.00668.x
PMCID: PMC2239301  PMID: 17973654
coat protein complex II; endoplasmic reticulum; multicopy suppressor; Saccharomyces cerevisiae; SEC24; vesicle formation
18.  The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome 
Molecular Biology of the Cell  2007;18(2):569-580.
The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.
doi:10.1091/mbc.E06-07-0635
PMCID: PMC1783769  PMID: 17135287
19.  Endoplasmic Reticulum Quality Control of Unassembled Iron Transporter Depends on Rer1p-mediated Retrieval from the Golgi 
Molecular Biology of the Cell  2004;15(3):1417-1424.
Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.
doi:10.1091/mbc.E03-10-0765
PMCID: PMC363159  PMID: 14699055
20.  Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane 
The Journal of Cell Biology  2003;161(6):1117-1131.
It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.
doi:10.1083/jcb.200303088
PMCID: PMC2172991  PMID: 12810702
ERG6; Tat2p; raft; multivesicular body; ubiquitin
21.  Rer1p, a Retrieval Receptor for ER Membrane Proteins, Recognizes Transmembrane Domains in Multiple Modes 
Molecular Biology of the Cell  2003;14(9):3605-3616.
The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Δrer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.
doi:10.1091/mbc.E02-12-0777
PMCID: PMC196553  PMID: 12972550
22.  Oligomerization of a Cargo Receptor Directs Protein Sorting into COPII-coated Transport Vesicles 
Molecular Biology of the Cell  2003;14(7):3055-3063.
Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII). The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors. Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Exit of Emp46p from the ER was saturable and dependent on the expression level of Emp47p. Emp46p binding to Emp47p occurs in the ER through the coiled-coil region in the luminal domains of both Emp47p and Emp46p, and dissociation occurs in the Golgi. Further, this coiled-coil region is also required for Emp47p to form an oligomeric complex of itself in the ER, which is essential for exit of Emp47p from the ER. Our results suggest that Emp47p is a receptor protein for Emp46p that allows for the selective transport of this protein, and this event involves receptor oligomerization.
doi:10.1091/mbc.E03-02-0115
PMCID: PMC165697  PMID: 12857885
23.  Cdc50p, a Conserved Endosomal Membrane Protein, Controls Polarized Growth in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2003;14(2):730-747.
During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.
doi:10.1091/mbc.E02-06-0314
PMCID: PMC150004  PMID: 12589066
24.  Ubiquitin Ligase Activities of Bombyx mori Nucleopolyhedrovirus RING Finger Proteins 
Journal of Virology  2003;77(2):923-930.
The genome of Bombyx mori nucleopolyhedrovirus (BmNPV) is predicted to contain six RING finger proteins: IAP1, ORF35, IAP2, CG30, IE2, and PE38. Several other members of the RING finger family have recently been shown to have the ubiquitin-ligase (E3) activity. We thus examined whether BmNPV RING finger proteins have the E3 activity. In vitro ubiquitination assay with the rabbit reticulocyte lysates and BmNPV RING finger proteins fused with maltose-binding protein (MBP) showed that four of them (IAP2, IE2, PE38, and CG30) were polyubiquitinated in the presence of zinc ion. Furthermore, MBP-IAP2, MBP-IE2, and MBP-PE38 were able to reconstitute ubiquitination activity in cooperation with the Ubc4/5 subfamily of ubiquitin-conjugating enzymes. Mutational analysis also showed that ubiquitination activity of MBP-IAP2, MBP-IE2, and MBP-PE38 were dependent on their RING finger motif. Therefore, these results suggest that IAP2, IE2, and PE38 may function as E3 enzymes during BmNPV infection.
doi:10.1128/JVI.77.2.923-930.2003
PMCID: PMC140854  PMID: 12502808
25.  Emp47p and Its Close Homolog Emp46p Have a Tyrosine-containing Endoplasmic Reticulum Exit Signal and Function in Glycoprotein Secretion in Saccharomyces cerevisiae 
Molecular Biology of the Cell  2002;13(7):2518-2532.
The yeast open reading frame YLR080w/EMP46 encodes a homolog of the Golgi protein Emp47p. These two proteins are 45% identical and have a single transmembrane domain in their C-terminal regions and a carbohydrate recognition domain signature in the N-terminal region. The C-terminal tail of Emp46p includes a dilysine signal. This protein is localized to Golgi membranes at steady state by subcellular fractionation and green fluorescent protein labeling. On block of forward transport in sec12-4 cells, redistribution of Emp46p from the Golgi to the endoplasmic reticulum is observed. These localization features are similar to those previously reported for Emp47p. In addition, mutagenesis of the C-terminal region identified a tyrosine-containing motif as a critical determinant of the Golgi-localization and interaction with both COPI and COPII components. Similar motifs are also observed in the C-terminal tail of Emp47p and other mammalian homologs. Disruption of Emp47p displays a growth defect at a high temperature or on Ca2+-containing medium, which is rescued by overexpression of Emp46p, suggesting a partially overlapping function between Emp46p and Emp47p. In addition, we found that the disruption of both Emp46p and Emp47p show a marked defect in the secretion of a subset of glycoproteins. Analysis of the C-terminal mutants for Ca2+ sensitivity revealed that the forward transport of Emp46/47p is essential for their function, whereas the retrograde transport is not. We propose that Emp46p and Emp47p are required for the export of specific glycoprotein cargo from the endoplasmic reticulum.
doi:10.1091/mbc.E02-01-0027
PMCID: PMC117331  PMID: 12134087

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