SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims: Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.<p></p> Methods: Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.<p></p> Key Results: VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.<p></p> Conclusions: These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in <i>Arabidopsis</i>

Background&lt;p&gt;&lt;/p&gt; The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell’s endomembrane, cytoskeletal and signal transduction apparatuses.&lt;p&gt;&lt;/p&gt; Results&lt;p&gt;&lt;/p&gt; In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic “unmasking” of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling.&lt;p&gt;&lt;/p&gt; Conclusions&lt;p&gt;&lt;/p&gt; Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism

Motion of the Zinc Ions in Catalysis by a Dizinc Metallo-β-Lactamase

We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-β-lactamase (MβL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MβL move away from each other, by ∼0.3 Å after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 Å. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 Å, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with nitrocefin. This is the first direct structural information available on early stage dizinc metallo-β-lactamase catalysis

A Five-coordinate Metal Center in Co(II)-substituted VanX

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of ∼100 m–1 cm–1, which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M = |±½〉, E/D = 0.14, greal(x,y) = 2.37, and grealS(z) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at ∼ 2.0 Å. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75–84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate d-Ala-d-Ala is also five-coordinate

Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from \u3cem\u3eBacillus anthracis\u3c/em\u3e

In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs

Sequential Binding of Cobalt(II) to Metallo-β-lactamase CcrA

In an effort to probe Co(II) binding to metallo-β-lactamase CcrA, EPR, EXAFS, and 1H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). 1H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn1 site in the enzyme

Structure and Metal Binding Properties of ZnuA, a Periplasmic Zinc Transporter from \u3cem\u3eEscherichia coli\u3c/em\u3e

ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated K d \u3c 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer