Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.

Virus-induced cytoplasmic inclusion bodies (referred to as virus replication complexes [VRCs]) consisting of virus and host components are observed in plant cells infected with tobacco mosaic virus, but the components that modulate their form and function are not fully understood. Here, we show that the tobacco mosaic virus 126-kD protein fused with green fluorescent protein formed cytoplasmic bodies (126-bodies) in the absence of other viral components. Using mutant 126-kD:green fluorescent fusion proteins and viral constructs expressing the corresponding mutant 126-kD proteins, it was determined that the size of the 126-bodies and the corresponding VRCs changed in synchrony for each 126-kD protein mutation tested. Through colabeling experiments, we observed the coalignment and intracellular trafficking of 126-bodies and, regardless of size, VRCs, along microfilaments (MFs). Disruption of MFs with MF-depolymerizing agents or through virus-induced gene silencing compromised the intracellular trafficking of the 126-bodies and VRCs and virus cell-to-cell movement, but did not decrease virus accumulation to levels that would affect virus movement or prevent VRC formation. Our results indicate that (1) the 126-kD protein modulates VRC size and traffics along MFs in cells; (2) VRCs traffic along MFs in cells, possibly through an interaction with the 126-kD protein, and the negative effect of MF antagonists on 126-body and VRC intracellular movement and virus cellto-cell movement correlates with the disruption of this association; and (3) virus movement was not correlated with VRC size.

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the V o sector, attached to a complex of peripheral membrane proteins, the V 1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V 1 subunits and the 100-kDa V o subunit associate within 3-5 min, followed by addition of other V o subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V 1 and V o sectors that form without any apparent V 1 V o subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V 1 subunits and the 100-kDa V o subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa V o subunit when the temperature block is reversed. We propose that assembly of the yeast VATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V 1 and V o subunits and an independent assembly pathway requiring full assembly of V 1 and V o sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of V o subunits during or after exit from the endoplasmic reticulum.Vacuolar proton-translocating ATPases (V-ATPases) 1 are highly conserved proton pumps found in all eukaryotic cells (reviewed in Ref. 1). V-type ATPases couple hydrolysis of cytoplasmic ATP to transport of protons from the cytosol into internal organelles or, in certain cells, across the plasma membrane. The catalytic sites for ATP hydrolysis reside in a peripheral complex of subunits called the V 1 sector of the enzyme, and the proton pore appears to be contained within a complex of integral membrane and tightly bound peripheral subunits called the V o sector. V-ATPases have been implicated in constitutive physiological processes ranging from protein sorting to pH and calcium homeostasis to activation of lysosomal proteases (reviewed in Refs. 1-3).The yeast V-type ATPase is composed of at least 13 different subunits, which have been identified by a combination of genetic and biochemical techniques (1, 4, 5). The V 1 sector of the yeast vacuolar ATPase is composed of a 69-kDa catalytic subunit, a 60-kDa subunit that appears to play a regulatory role, and six other peripheral subunits of relative molecular masses 54, 42, 32, 27, 14, and 13 kDa. The V o sector of the yeast enzyme consists of a 100-kDa integral membrane subunit, a tightly associated peripheral subunit of 36 kDa, and a trio of proteolipid subunits of 23, 17, and 1...

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H 2 O 2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage. Reactive oxygen species (ROS)2 can be formed as by-products in all oxygenic organisms during aerobic metabolism (1). In higher plants, chloroplasts and mitochondria are two major organelles that contribute to production of reactive oxygen species during photosynthesis and carbon metabolism (2, 3). In addition, plants actively generate ROS as signals in response to environmental stresses (3-6). However, because of the cytotoxic and extremely reactive nature of ROS, they can cause wide ranging damage to macromolecules (1, 7-9). To overcome such oxidative damage and control signaling events, plants have orchestrated an elaborate antioxidant network (4).Of those antioxidant systems, the physiological roles of thioredoxins have been intensively studied (10), whereas those of Grxs have not been fully defined (11,12). Grxs are ubiquitous small heat-stable disulfide oxidoreductases, which are conserved in both prokaryotes and eukaryotes (11, 13). Through an active motif, namely the conserved CPYC sequence (a dithiol Grx), they catalyze the reduction of protein disulfides and of GSHprotein mixed disulfides via a dithiol or monothiol mechanism (14, 15). In bacterial, yeast, and mammalian cells, dithiol Grxs appear to be involved in many cellular processes and play an important role in protecting cells against oxidative stresses (16 -18).Besides the dithiol Grxs, a new group of monothiol Grxs has recently been identified in yeast (Grx3, -4, and -5) and bacteria (Grx4) that have a single cysteine residue in the putative active motif (19,20). Yeast Grx5 encodes...

Global environmental temperature changes threaten innumerable plant species. Although various signaling networks regulate plant responses to temperature fluctuations, the mechanisms unifying these diverse processes are largely unknown. Here, we demonstrate that an Arabidopsis monothiol glutaredoxin, AtGRXS17 (At4g04950), plays a critical role in redox homeostasis and hormone perception to mediate temperature-dependent postembryonic growth. AtGRXS17 expression was induced by elevated temperatures. Lines altered in AtGRXS17 expression were hypersensitive to elevated temperatures and phenocopied mutants altered in the perception of the phytohormone auxin. We show that auxin sensitivity and polar auxin transport were perturbed in these mutants, whereas auxin biosynthesis was not altered. In addition, atgrxs17 plants displayed phenotypes consistent with defects in proliferation and/or cell cycle control while accumulating higher levels of reactive oxygen species and cellular membrane damage under high temperature. Together, our findings provide a nexus between reactive oxygen species homeostasis, auxin signaling, and temperature responses.

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.

Alzheimer’s disease (AD), one of the most dreaded neurodegenerative disorders, is characterized by cortical and cerebrovascular Aβ (amyloid β peptide) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss. Increased bone fracture rates and reduced bone density are commonly observed in patients with AD, suggesting a common denominator(s) between both disorders. However, very few studies are available that have addressed this issue. Here, we present evidence for a function of amyloid precursor protein (APP) and Aβ in regulating osteoclast (OC) differentiation in vitro and in vivo. Tg2576 mice, which expresses Swedish mutation of APP (APPswe) under the control of prion promoter (1,2), exhibit biphasic effects on OC activation, with an increase of OC in younger mice (< 4 month old), but a decrease in older Tg2576 mice (> 4 month old). The increase of OC in young Tg2576 mice appears to be mediated by Aβ oligomers and RAGE (receptor for advanced glycation end products) expression in BMMs. However, the decrease of OC formation and activity in older Tg2576 mice may be due to the increase of sRAGE in aged Tg2576 mice, an inhibitor of RANKL induced osteoclastogenesis. These results suggest an unexpected function of APPswe/Aβ, reveal a mechanism underlying altered bone remodeling in AD patients, and implicate APP/Aβ and RAGE as common denominators for both AD and osteoporosis.

SummaryWhile various signalling networks regulate plant responses to heat stress, the mechanisms regulating and unifying these diverse biological processes are largely unknown. Our previous studies indicate that the Arabidopsis monothiol glutaredoxin, AtGRXS17, is crucial for temperature-dependent postembryonic growth in Arabidopsis. In the present study, we further demonstrate that AtGRXS17 has conserved functions in anti-oxidative stress and thermotolerance in both yeast and plants. In yeast, AtGRXS17 co-localized with yeast ScGrx3 in the nucleus and suppressed the sensitivity of yeast grx3grx4 double-mutant cells to oxidative stress and heat shock. In plants, GFP-AtGRXS17 fusion proteins initially localized in the cytoplasm and the nuclear envelope but migrated to the nucleus during heat stress. Ectopic expression of AtGRXS17 in tomato plants minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane systems under heat stress. This enhanced thermotolerance correlated with increased catalase (CAT) enzyme activity and reduced H 2 O 2 accumulation in AtGRXS17-expressing tomatoes. Furthermore, during heat stress, expression of the heat shock transcription factor (HSF) and heat shock protein (HSP) genes was up-regulated in AtGRXS17-expressing transgenic plants compared with wild-type controls. Thus, these findings suggest a specific protective role of a redox protein against temperature stress and provide a genetic engineering strategy to improve crop thermotolerance.