75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

133 The Road Less Travelled: Investigating The Multi-Step Targeting Pathway of Tic40
Joanna Tripp, Kenneth Keegstra, John Froehlich
DOE-MSU Plant Research Laboratory, Michigan State University, East Lansing MI, 48823 (USA)
Proteins destined for the chloroplastic inner envelope membrane appear to use the general import pathway, but
then get diverted to the inner envelope membrane by an unknown process. The pathways and mechanisms for targeting
proteins to the chloroplastic inner envelope membrane are poorly characterized. Based on analogy with mitochondria,
where targeting to the inner membrane has been extensively studied, two hypotheses are possible. First is the stop transfer
hypothesis whereby transport across the inner envelope membrane is halted, leading to insertion into the inner envelope
membrane during the import process. The second hypothesis, sometimes called the conservative sorting hypothesis,
predicts that import into the stroma is completed before insertion into the inner envelope membrane occurs as a discrete
second step.
Tic40 is part of the protein import apparatus located at the inner envelope membrane of chloroplasts. It is anchored
in the inner envelope membrane by a single N-terminal transmembrane domain and has a topology in which the bulk of
the C-terminal domain is orientated toward the stroma. In vitro studies indicate that the targeting of Tic40 to the inner
envelope membrane involves two steps. Using an in vitro import assay, we have shown that in the first step, Tic40 is
initially transported across the inner envelope membrane into the stroma. The resulting Tic40 stromal intermediate is
subsequently retargeted to the inner envelope membrane by an unknown mechanism. The sorting of Tic40 requires a
bipartite transit peptide, which is first cleaved by the stromal processing peptidase (SPP) thus generating a soluble Tic40
stromal intermediate (iTic40). iTic40 is further processed by a second peptidase, possibly a Type I Signal peptidase,
which generates its mature form (mTic40). Using deletion mutants, we are investigating and characterizing the targeting
determinants of Tic40 involved in this multi-step targeting pathway. Presently, we have identified a sequence motif N-
terminal of the transmembrane domain which appears to prevent arrest of Tic40 at the inner envelope membrane and
allows complete transport of Tic40 into the stroma. Likewise, we are continuing to investigate and identify additional
targeting determinants that are required for reinsertion of iTic40 into the inner envelope membrane. Finally, by examining
the multi-step insertion of Tic40 into the inner envelope, we envision that Tic40 may be a good ‘model’ protein that will
allows us to investigate in greater detail the ‘conservative sorting pathway’ within chloroplasts.
134 Arabidopsis microtubule-associated protein SPIRAL2 affects microtubule dynamics
Maki Yao, Tsubasa Shoji, Takashi Hashimoto
Nara Institute of Science and Technology(NAIST), Nara, Japan
In plant cells, various microtubule-associated proteins (MAPs) regulate many microtubule (MT) functions such
as ordered array formation in interphase and phragmoplast development. However, exact biochemical functions of MAPs
are largely unknown. Arabidopsis SPIRAL2/TORTIFOLIA (SPR2) protein is a plant-specific MAP involved in the
regulation of cortical MT arrays which dictate the direction of cell expansion. SPR2 localizes to MTs in vivo, and the
null mutation of SPR2 causes a shallow left-handed helical array in cortical MTs and a right-handed helical growth in
longitudinally growing organs such as hypocotyls, petioles and flowers. SPR2 has HEAT repeat motifs at the N-terminal
region and highly conserved region among SPR2 homologs at the C-terminus. In MT binding assays in vitro, nonoverlapping
N- and C-terminal regions of recombinant SPR2 directly bound to taxol-stabilized MTs. We also examined
the effect of SPR2 on the dynamics of MT by dark-field microscopy using the recombinant proteins of full-length and
N-terminal SPR2. Results will be presented.