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preproteins are prone to aggregation or
misfolding in the cytosol, thus preventing
efficient import.
Therefore, in this research project we are
investigating the molecular and regulatory
role of cytosolic chaperones as well as the
posttranslational modification of preproteins
during protein sorting and translocation
into chloroplasts, mitochondria and the
endoplasmic reticulum. Phosphorylation
of chloroplast preproteins by cytosolic
kinases ensures efficient protein import
during chloroplast biogenesis. Chaperoneassociated preproteins can be initially
recognised by membrane receptor proteins
and are subsequently transferred to the
import channels.
What sparked your interest in plant
sciences and can you summarise your
ultimate ambitions in this arena?
Why were Arabidopsis, pea and
wheat chosen as the model systems
for your research?
Plants are fascinating organisms and provide
the prerequisite for all life on Earth through
their ability to convert sunlight into chemical
energy. For this reason, their cells contain
an extra organelle – the chloroplast – which
makes the sorting of proteins synthesised
in the cytosol especially challenging. My
research aims at identifying the underlying
molecular mechanisms that ensure efficient
biogenesis and maintenance of the plant cell.
Arabidopsis is uniquely suited as a plant model
organism since it has a fairly short lifecycle
and the entire genome was sequenced over
a decade ago. A large collection of mutants
is available and genetic modification is
possible. However, Arabidopsis is not a big
plant in terms of biomass, and isolating large
amounts of subcellular compartments and
membranes is therefore difficult. For this
reason, we are using pea, which can be grown
quickly and produces biomass for biochemical
experiments. A lysate, which we isolate
from wheat germs, can be used as an in vitro
protein translation system for exogenously
added RNA.
Can you detail the key aims of your current
research project ‘HSP90 function in
preprotein import into organelles’?
Our research investigates posttranslational
processes during the transport of preproteins
to cellular compartments in the cytosol.
Protein transport in the plant cell is highly
dynamic and regulated, since it is essential
for the integration of organelles into the
cellular network. Most organellar proteins
are synthesised in the cytosol and must be
transported to their destined organelles
in an import competent state. However,
Could you describe some of the main
methods used in your research?
In order to identify and analyse the
components involved in posttranslational
protein import we are using molecular,
biochemical and cell biological techniques.
For example, we fuse our proteins of interest
to fluorescent proteins and express these
DR SERENA SCHWENKERT
Dr Serena Schwenkert provides an insight into the motivations and mission driving her
current research on posttranslational processes during the transport of preproteins to cellular
compartments within plants, and the broad range of methods facilitating this work
in living cells. This allows us to visualise
their localisation within the cell. To take a
closer look at protein-protein interactions,
we can fuse split fluorescent proteins to
the proteins we want to investigate. If the
two proteins are expressed in the cell and
thereby come into very close proximity, the
split fluorescent proteins can interact and we
can observe the reconstituted fluorescence.
Moreover, we can genetically modify the
proteins on specific sites, thus allowing us to
investigate the function of individual amino
acids or protein regions. With our broad
range of methods we aim to analyse protein
transport on both an in vivo and in vitro level.
Your research group comprises two PhD
students. To what extent are you involved
in nurturing the next generation of
researchers in your field?
My work provides a great opportunity to
work with highly motivated students not
only at the PhD level, but also during their
Master and Bachelor studies. It is a pleasure
to catch their excitement and interest in
plant sciences in combination with modern
biochemical techniques. We recently
established a practical course in which the
students learn to purify and characterise
proteins using a chromatography system.
Do you think that there is enough
research conducted within the field of
plant sciences, specifically in terms of
protein interactions?
Over the past years, methods to study
protein interactions have significantly
improved. However, focus should be on
techniques that allow for the monitoring
of such interactions in vivo. This remains
a challenge, since most of the techniques
interfere with the natural status of the
proteins, for example by adding large
‘tags’ to the proteins, or by the need
to solubilise proteins from their lipid
membrane surroundings.
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DR SERENA SCHWENKERT
To the root of protein transport
By using in vitro and in vivo methods to study Arabidopsis, pea and wheat model systems, researchers
at the Department of Biology at Ludwig Maximilian University of Munich hope to reveal insights
into posttranslational processes in plants, and have already unearthed important findings
FROM PROVIDING OXYGEN to producing
basic foodstuffs, plants make life on Earth
possible for humans and animals. With
more than 300,000 species, plants are
vital to ecologies across the globe – within
rainforests, deserts, the oceans and even in
cities, where there are increasing efforts to
rejuvenate their presence. The importance of
plants for human survival is well understood,
but scientists still have many questions
about how plants have successfully inhabited
the Earth for millennia.
Shedding light on how plants function at
their most basic level – the cellular – is key
to establishing a complete understanding of
plant biogenesis. Focusing on gaining greater
insight into posttranslational processes
during the transport of preproteins to cellular
compartments in the cytosol, Dr Serena
Schwenkert’s research group at Ludwig
Maximilian University of Munich is engaged in
the HSP90 function in preprotein import into
organelles project.
CHAPERONE PROTEINS
Heat shock protein 90 (HSP90) serves
an important function in maintaining the
homeostasis of proteins, and thus the survival
of plants. HSP90 was first isolated by removing
proteins from cells that were intentionally
stressed by heating or dehydrating, which led
scientists to observe that the cell’s proteins
began to denature as a result. HSP90 is also
essential in unstressed cells where it plays a
role in assisting folding, intracellular transport,
maintenance, and degradation of proteins, as
well as facilitating cell signalling.
Therefore, HSP90 is considered a chaperone
protein and works alongside helper proteins
called ‘co-chaperones’ in a complex sequential
cycle. “In the context of preprotein import,
HSP90 associates to freshly synthesised
preproteins to prevent aggregation with other
proteins in the cytosol and to mediate a first
contact with the translocon machineries,”
Schwenkert explains.
116INTERNATIONAL INNOVATION
A COMPLEX NETWORK
The main aim of the project is to understand
the mechanisms behind cytosolic factors,
including chaperones such as HSP90. These
factors bind to preproteins, a process that
expands the range of functions of a protein by
joining it with other biochemical functional
groups. Posttranslational modification is
another area of investigation for Schwenkert:
“We would like to understand the complex
network of protein interactions that
contribute to guide the preproteins efficiently
from their place of synthesis to their place of
function,” she reveals.
Furthermore, Schwenkert and her colleagues
are looking to shed light on how preproteins
are recognised at the organellar surfaces by
analysing the direct and indirect interaction
of preproteins with membrane receptor
proteins, and their transfer to the respective
membrane pores.
DUAL APPROACH
Despite the use of both in vitro and in vivo
methods in Schwenkert’s research, it can
be very difficult to study the stages of preprotein passage within the plant cell after
translation in the cytosol. This is because the
steps are very short-lived, and in vitro systems
that have been used in the past to study
protein import utilise isolated organelles,
which are no longer surrounded by the
natural cytosol. Moreover, the conditions
found within the cell are naturally different
to those applied in studies, since researchers
use preproteins in excess when investigating
them. “Learning more will help us to better
understand the integration of organelles into
the cellular network and their dependence on
efficient sorting and targeting mechanisms of
preproteins,” Schwenkert predicts.
Specifically, in order to identify and analyse
the components involved in posttranslational
protein import, Schwenkert’s lab is using
Arabidopsis, pea and wheat model systems. The
researchers created a homemade wheatgerm
extract that allows them to simulate the
cytosolic conditions that take place naturally
within the plant cell. Using this unique extract
also allows for the labelling and visualisation
of specific proteins. “In addition to this, we are
working with recombinant proteins produced
in bacteria, which we can modify and thus
investigate the functions of individual amino
acids or defined protein regions,” Schwenkert
elucidates. “Moreover, we are using transgenic
approaches to work with modified plants
either hampered in the function of specific
proteins or expressing proteins ectopically.”
SUCCESS TO DATE
Thus far, Schwenkert’s approach has seen
success, with the group having unearthed
several key findings, including identifying
several components of the HSP90 machinery,
which can also be linked to chloroplast
preprotein import. In one study, a large set of
wheat germ-translated chloroplast preproteins
were analysed with respect to chaperone
Wild type (right) and kinase mutant plants (left) during
greening (top) and under normal growth conditions
(bottom) (also see Lamberti et al. 2011).
Model of chaperone bound preproteins
recognised by TPR receptors.
INTELLIGENCE
HSP90 FUNCTION IN PREPROTEIN
IMPORT INTO ORGANELLES
OBJECTIVES
• To identify and analyse the components
involved in posttranslational protein
import using molecular, biochemical and
cell biological techniques
• To specify the role of the HSP90
chaperone in preprotein targeting
binding. This revealed that 14–3–3 proteins or
HSP90-containing preprotein complexes are a
common feature in posttranslational protein
transport. As a result of this, the lab was able
to reveal a tool for the investigation of HSP90
client protein association – an extensive class of
preproteins as HSP90 substrates.
Schwenkert’s research is also looking into AtTPR7
– another membrane receptor facilitating
protein-protein interaction – and has already
uncovered its potential role in posttranslational
protein import. “We have identified a novel
receptor protein in the membrane of the
endoplasmic reticulum, which we propose to
function in HSP90-mediated import into this
cellular compartment,” she reveals. The findings
from the study have highlighted the important
function of AtTPR7 in the posttranslational
protein import into the endoplasmic reticulum,
which suggests that plants contain a mechanism
for posttranslational sorting between the
endoplasmic reticulum, mitochondria and
chloroplasts in cells.
Additionally, progress has been made in better
understanding the function of chloroplast
preprotein phosphorylation. “Our data could
Fluorescent labeled plant organelles in isolated
protoplasts. Chloroplast (autofluorescence in red),
endoplasmic reticulum (yellow), mitochondria (blue),
outer membrane of the chloroplast (green).
show that it has a specific function in the very
early stages of chloroplast development,”
Schwenkert highlights. “During the biogenesis
of chloroplasts in expanding leaves the demand
for imported proteins is especially high and
phosphorylation enhances the import of
specific preproteins.”
FILLING THE GAPS
Although Schwenkert’s lab has clearly made
significant strides in improving knowledge about
posttranslational processes during preprotein
transport, there are still a number of gaps in
understanding that she and her colleagues are
looking to fill. “We will investigate the protein
folding status of preproteins in the cytosol as
well as during their integration into protein
complexes within the organelles,” Schwenkert
reveals. “Moreover, we are aiming to unravel
control mechanism which prevents missorting
in the cell and how dual targeting of identical
proteins to several organelles is achieved.”
Moving forward, Schwenkert and her colleagues
hope to paint a complete picture of how proteins
travel from where they are synthesised in the
plant to their final place of function, improving
our understanding of the lifecycle of plants that
underpins our existence.
Fluorescent labelled endoplasmic reticulum (green) and
chloroplasts (red) in an entire tobacco leaf.
• To analyse the impact of
posttranslational phosphorylation of
preproteins on the import of preproteins
into chloroplasts
• To elucidate the composition and
functioning of the plant Sec translocon
in the endoplasmic reticulum
KEY COLLABORATORS
Professor Adina Breiman, Tel Aviv
University, Israel
Professor Elzbieta Glaser, University of
Stockholm, Sweden
Professor Johannes Buchner, TU
München
FUNDING
The German Research Foundation (DFG)
CONTACT
Dr Serena Schwenkert
Biozentrum der LMU München
Department Biologie I, Botanik
Großhadernerstr. 2-4
82152 Planegg-Martinsried
Germany
T +49 89 2180 74760
E [email protected]
DR SERENA SCHWENKERT began
working as a research group leader
alongside the chair of Plant Biochemistry
and Physiology, Professor Jürgen Soll,
having completed her PhD at Ludwig
Maximilian University of Munich in 2008.
Schwenkert’s work is currently funded by
the DFG within the collaborative research
centre ‘Control of protein function by
conformational switching’ (CRC 1035).
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