Content
2007
Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum
21.12.2007
Eur J Cell Biol.,
2008,
87,
237-249
published on 21.12.2007
The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated
DdKif3-592 motor (aa 1–592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0 mms -1 at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1–342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFPfusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to
both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than
animal kinesins.
A step dissected
29.11.2007
The motor protein kinesin ‘walks’ by alternately advancing its two motor structural domains. A cutting-edge, single-molecule fluorescencetechnique reveals further details of this stepping mechanism.
Suppose that walking required energy input in the form of, say, one Gummi bear for every step. In what position would you pop the next Gummi bear into your mouth? When one foot is firmly planted on the ground while the other is lifted up and poised next to it to be thrust
forward, or when both feet are on the ground spaced apart at step size? Mori et al. have addressed these questions, not in a person, but in a miniature, biological walking machine — the molecular motor protein kinesin.
A Grp on the Hsp90 Mechanism
26.10.2007
In a recent issue of Molecular Cell, Dollins et al. (2007) present the crystal structure of Grp94, which highlights the similarity between Grp94 and Hsp90 and provides insight into the resting state of Grp94 and potentially other Hsp90 family members.
The ATP-ase cycle of the endoplamic chaperone GRP94
09.10.2007
Grp94, the Hsp90 paralogue of the endoplasmic reticulum (ER), plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question whether Grp94 hydrolyses ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been
unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential
ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable to that of yeast Hsp90 with a kcat of 0.36 min-1 at 25°C. Kinetic and equilibrium constants of the partial
reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared to other Hsp90s investigated, with dissociation constants (KD) of about 4 μM for ADP, ATP and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, NECA) studied here bind with similar rate constants for association (0.2-0.3 x 106 M-1 s-1). Furthermore, there is a marked difference
to cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.
Protein transport in chloroplasts - targeting to the intermembrane space
01.10.2007
the FEBS Journal,
2007,
274,
5043–54
published on 01.10.2007
www.febsjournal.org
www.febsjournal.org
The import of proteins destined for the intermembrane space of chloroplasts has not been investigated in detail up to now. By investigating energy requirements and time courses, as well as performing competition experiments, we show that the two intermembrane space components Tic22 and MGD1 (E.C. 2.4.1.46) both engage the Toc machinery for crossing the outer envelope, whereas their pathways diverge thereafter. Although MGD1 appears to at least partly cross the inner envelope, Tic22 very likely reaches its mature form in the intermembrane space without involving stromal components. Thus, different pathways for intermembrane space targeting probably exist in chloroplasts.
