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Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba
J F Kelleher1, S J Atkinson, T D Pollard
1Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Abstract:
We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.
Insights
Researchers sequenced actin-related proteins (Arps) in Acanthamoeba, revealing distinct Arp2 and Arp3 families crucial for eukaryotic cell structure. These Arps may nucleate actin polymerization, functioning in the cell cortex.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Actin-related proteins (Arps) are essential components of the cytoskeleton.
- The profilin-binding complex in Acanthamoeba contains two Arps, Arp2 and Arp3.
- Understanding Arp structure and function is key to deciphering actin dynamics.
Purpose of the Study:
- To clone and sequence the two Arps in the Acanthamoeba profilin-binding complex.
- To perform phylogenetic analysis of Arps and their relationship to actin and other ATPases.
- To predict the function of Arp2 and Arp3 based on atomic models and cellular localization.
Main Methods:
- Gene cloning and DNA sequencing of Acanthamoeba Arps.
- Phylogenetic analysis of Arp sequences.
- Construction of atomic models based on sequence and actin structure.
- Cellular fractionation and protein concentration determination.
Main Results:
- Arp2 and Arp3 sequences are distinct and group into major families across eukaryotes.
- Arps form a larger family with conventional actins, separate from other ATPases.
- Atomic models suggest Arps bind ATP and cations; Arp2 has a profilin-binding site.
- Arps do not copolymerize with actin but may nucleate polymerization as a heterodimer.
- Acanthamoeba Arps are soluble, concentrated in the cell cortex, and present at substoichiometric levels to actin.
Conclusions:
- Arp2 and Arp3 represent conserved protein families with distinct evolutionary paths.
- The Arp heterodimer may function as a nucleus for actin polymerization.
- Arps are integral components of the Acanthamoeba cytoskeleton, particularly in the cell cortex.