144 research outputs found
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ATAD3 Proteins: Unique Mitochondrial Proteins Essential for Life in Diverse Eukaryotic Lineages
ATPase family AAA domain–containing 3 (ATAD3) proteins are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent in Fungi and Amoebozoa. These ∼600-amino acid proteins are anchored in the inner mitochondrial membrane and are essential in metazoans and Arabidopsis thaliana. ATAD3s comprise a C-terminal ATPases Associated with a variety of cellular Activities (AAA+) matrix domain and an ATAD3_N domain, which is located primarily in the inner membrane space but potentially extends to the cytosol to interact with the ER. Sequence and structural alignments indicate that ATAD3 proteins are most similar to classic chaperone unfoldases in the AAA+ family, suggesting that they operate in mitochondrial protein quality control. A. thaliana has four ATAD3 genes in two distinct clades that appear first in the seed plants, and both clades are essential for viability. The four genes are generally coordinately expressed, and transcripts are highest in growing apices and imbibed seeds. Plants with disrupted ATAD3 have reduced growth, aberrant mitochondrial morphology, diffuse nucleoids and reduced oxidative phosphorylation complex I. These and other pleiotropic phenotypes are also observed in ATAD3 mutants in metazoans. Here, we discuss the distribution of ATAD3 proteins as they have evolved in the plant kingdom, their unique structure, what we know about their function in plants and the challenges in determining their essential roles in mitochondria
The Chloroplast Small Heat-Shock Protein Oligomer Is Not Phosphorylated and Does Not Dissociate during Heat Stress in Vivo
Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes
Small heat shock proteins (sHSPs) are dynamic oligomeric
proteins that bind unfolding proteins and protect
them from irreversible aggregation. This binding results
in the formation of sHSP-substrate complexes from
which substrate can later be refolded. Interactions between
sHSP and substrate in sHSP-substrate complexes
and the mechanism by which substrate is transferred to
ATP-dependent chaperones for refolding are poorly defined.
We have established C-terminal affinity-tagged
sHSPs from a eukaryote (pea HSP18.1) and a prokaryote
(Synechocystis HSP16.6) as tools to investigate these issues.
We demonstrate that sHSP subunit exchange for
HSP18.1 and HSP16.6 is temperature-dependent and
rapid at the optimal growth temperature for the organism
of origin. Increasing the ratio of sHSP to substrate
during substrate denaturation decreased sHSP-substrate
complex size, and accordingly, addition of substrate
to pre-formed sHSP-substrate complexes increased
complex size. However, the size of pre-formed
sHSP-substrate complexes could not be reduced by addition
of more sHSP, and substrate could not be observed
to transfer to added sHSP, although added sHSP
subunits continued to exchange with subunits in sHSPsubstrate
complexes. Thus, although some number of
sHSP subunits within complexes remain dynamic and
may be important for complex structure/solubility, association
of substrate with the sHSP does not appear to be
similarly dynamic. These observations are consistent
with a model in which ATP-dependent chaperones associate
directly with sHSP-bound substrate to initiate
refolding
Auxin efflux controls orderly nucellar degeneration and expansion of the female gametophyte in Arabidopsis
The nucellus tissue in flowering plants provides nutrition for the development of the female gametophyte (FG) and young embryo. The nucellus degenerates as the FG develops, but the mechanism controlling the coupled process of nucellar degeneration and FG expansion remains largely unknown. The degeneration process of the nucellus and spatiotemporal auxin distribution in the developing ovule before fertilization were investigated in Arabidopsis thaliana. Nucellar degeneration before fertilization occurs through vacuolar cell death and in an ordered degeneration fashion. This sequential nucellar degeneration is controlled by the signalling molecule auxin. Auxin efflux plays the core role in precisely controlling the spatiotemporal pattern of auxin distribution in the nucellus surrounding the FG. The auxin efflux carrier PIN1 transports maternal auxin into the nucellus while PIN3/PIN4/PIN7 further delivers auxin to degenerating nucellar cells and concurrently controls FG central vacuole expansion. Notably, auxin concentration and auxin efflux are controlled by the maternal tissues, acting as a key communication from maternal to filial tissue
Interrogating Plant Cell Culture Library for Novel Antimicrobial Agents
The Plant Cell Culture Library (PCCL) at UMass Amherst contains more than 2,200 live plant cell cultures, representing diverse plant species from around the world. The availability of this collection offers a rich resource for us to discover bioactive phytochemicals and uncover their mechanisms of action. Using data-mining surveys of bioactive plant extracts, I have organized subsets of PCCL cell lines that are likely to possess antifungal, antibacterial, antiviral, anthelmintic, anti-trypanosomal, or anticancer properties, which prove to be useful when deciding which species to screen first against a specific pathogen. Another distinct advantage of using the live plant cells in this research is the ability to stimulate the biosynthesis of pathogen-specific phytochemicals upon simulation of an attack (elicitation) by the microorganism in question. This could be accomplished by pathogen homogenates or plant hormones responsible for mounting defenses to infection.
Over the past six months, I have been working to optimize elicitation, lysis, and extraction conditions for obtaining high-throughput screening materials to be used against variable pathogens. Equipped with crude extracts from appropriately elicited cells, I am collaborating with a multidisciplinary team of UMass scientists to develop and implement high-throughput screening protocols for profiling a large number of plant-derived materials against various pathogens. Recently, I have screened a small pool (40) of extracts derived from cell lines with predicted anti-fungal properties against the highly resistant strain of fungus Fusarium oxysporum, one of the causal agents of an opportunistic infection often seen in immunocompromised patients known as fusariosis. Gratifyingly, I have found several plant species that produced specialized metabolites with better antifungal activity than the leading antibiotic against F. oxysporum, Amphotericin B, validating this line of antimicrobial research. We are also actively reaching out to other academic labs partners to form partnerships in diverse antimicrobial research venues
Cloning, Sequence Analysis, and Expression of a cDNA Encoding a Plastid-Localized Heat Shock Protein in Maize
The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions
The small heat shock proteins (sHSPs) are a ubiquitous
class of ATP-independent chaperones believed to
prevent irreversible protein aggregation and to facilitate
subsequent protein renaturation in cooperation
with ATP-dependent chaperones. Although sHSP chaperone
activity has been studied extensively in vitro, understanding
the mechanism of sHSP function requires
identification of proteins that are sHSP substrates in
vivo. We have used both immunoprecipitation and affinity
chromatography to recover 42 proteins that specifically
interact with Synechocystis Hsp16.6 in vivo during
heat treatment. These proteins can all be released from
Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones
and are heat-labile. Thirteen of the putative
substrate proteins were identified by mass spectrometry
and reveal the potential for sHSPs to protect cellular
functions as diverse as transcription, translation, cell
signaling, and secondary metabolism. One of the putative
substrates, serine esterase, was purified and tested
directly for interaction with purified Hsp16.6. Hsp16.6
effectively formed soluble complexes with serine esterase
in a heat-dependent fashion, thereby preventing formation
of insoluble serine esterase aggregates. These
data offer critical insights into the characteristics of
native sHSP substrates and extend and provide in vivo
support for the chaperone model of sHSP function
The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions
The small heat shock proteins (sHSPs) are a ubiquitous
class of ATP-independent chaperones believed to
prevent irreversible protein aggregation and to facilitate
subsequent protein renaturation in cooperation
with ATP-dependent chaperones. Although sHSP chaperone
activity has been studied extensively in vitro, understanding
the mechanism of sHSP function requires
identification of proteins that are sHSP substrates in
vivo. We have used both immunoprecipitation and affinity
chromatography to recover 42 proteins that specifically
interact with Synechocystis Hsp16.6 in vivo during
heat treatment. These proteins can all be released from
Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones
and are heat-labile. Thirteen of the putative
substrate proteins were identified by mass spectrometry
and reveal the potential for sHSPs to protect cellular
functions as diverse as transcription, translation, cell
signaling, and secondary metabolism. One of the putative
substrates, serine esterase, was purified and tested
directly for interaction with purified Hsp16.6. Hsp16.6
effectively formed soluble complexes with serine esterase
in a heat-dependent fashion, thereby preventing formation
of insoluble serine esterase aggregates. These
data offer critical insights into the characteristics of
native sHSP substrates and extend and provide in vivo
support for the chaperone model of sHSP function
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