Cytosolic proteins from tobacco pollen tubes that crosslink microtubules and actin filaments in vitro are metabolic enzymes

Abstract

In plant cells, many processes require cooperative action of both microtubules and actin filaments, but proteins mediating interactions between these cytoskeletal members are mostly undiscovered. Here, we attempt to identify such proteins by affinity purification. Cytosol from Nicotiana tabacum (tobacco) pollen tubes was incubated first with actin filaments, and then proteins eluted from the actin were incubated with microtubules, and finally those microtubule-binding proteins were pooled in an active fraction. This fraction bundled actin filaments but not microtubules. However, when the fraction was added to both actin and microtubules, large bundles resulted, containing both polymers, regardless of the order of addition of components. Similar results were obtained when the order of affinity purification was reversed. The four most abundant bands from the fractions were identified from peptide fragments analyzed by mass spectrometry. The same four proteins were identified regardless of the order of affinity purification. The proteins are: homocysteine methyltransferase, phosphofructokinase, pyruvate decarboxylase, and glucan protein synthase (reversibly glycosylated protein). These results suggest the importance of structuring metabolism within the confines of the pollen tube cytoplasm.

Organelle trafficking, the cytoskeleton, and pollen tube growth

“…The degree of overlap and alignment is not known although several datasets suggest that the two systems interact. Optical and electron microscopy images show the close alignment between actin filaments and microtubules (Pierson et al ; Lancelle and Hepler , 1992), while biochemical data reveal the existence of proteins that bridge the two systems (Huang et al ; Romagnoli et al ; Wang et al ). Furthermore, the selective depolymerization of one of the two systems significantly impacts the other (Poulter et al ).…”

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

“…We cannot exclude the possibility that such interactions involve metabolic enzymes thereby taking cytoskeletal sensing to a very high level of integration. Some support for this possibility is given by the findings that (1) a fraction from tobacco pollen tubes containing PFK, homocysteine methyltransferase, pyruvate decarboxylase, and glucan protein synthase promoted the bundling of actin microfilaments and the interaction of these microfilaments with microtubules [34] and (2) in apple pollen, the binding of actin filaments to a transglutaminase (which catalyses the reaction between acyl acceptor glutamyl residues and amine donors) led to the aggregation of actin and to a similar aggregation using tubulin [35]. That said, the binding of metabolic enzymes to both microfilaments and MTs at the same time may be rare given that the binding of enzymes to the different cytoskeletal filaments is frequently isoform-specific and can vary with the activity state of the enzyme.…”

Fueled by microtubules: Does tubulin dimer/polymer partitioning regulate intracellular metabolism?

“…were reported in animal cells, but evidence for MT binding has also been presented in plants [e.g., Chuong et al, 2004;Romagnoli et al, 2010] and the yeast Saccharomyces cerevisiae [Gotz et al, 1999].…”