Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5

Abstract

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicatedmodelofhumanizedyeast.HumanTauwasreadilyphosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3␤ and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, ⌬mds1 and ⌬pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

“…Constructs and protocols for the expression of the longest human Tau isoform (Tau-2N/4R; 441 amino acids (aa)) were as described previously (17)(18)(19). The Y18E point mutation was introduced in Tau using the QuikChange II XL site-directed mutagenesis kit (Agilent, Diegem, Belgium) using the forward and reverse mutagenesis primers 5Ј-TCACGCTGGGACGGAGGGGTTGGGGG-ACA-3Ј and 5Ј-TGTCCCCCAACCCCTCCGTCCCAGCG-TGA-3Ј.…”

“…For immunizations, the purified Tau was activated with glutaraldehyde and coupled to keyhole limpet hemocyanin via a two-step enamine covalent coupling to generate keyhole limpet hemocyanin-coupled Tau. For the dephosphorylation studies, purified Tau extracted from the ⌬pho85 strain was treated with shrimp alkaline phosphatase (AP) (Roche Applied Science) according to the manufacturer's recommendations (17,18).…”

“…The expression of human Tau (longest isoform, 2N/4R; 441 aa) in yeast has been characterized extensively before, thereby showing robust traits found in AD (17)(18)(19). In this study, we focused mainly on expression of Tau in the pho85⌬ strain, which lacks the yeast orthologue of Cdk5 and displays extensive hyperphosphorylation of protein Tau and higher levels of Sarkosyl-insoluble Tau as compared with the BY4741 wild type strain (18).…”

“…All antibodies used for detection of Tau or reference proteins are listed in Table 3. Dephosphorylation of Tau in yeast extracts was performed using alkaline phosphatase as described previously (17). For immunohistochemistry, we used free floating coronal sections of 40 m for THY-Tau22 transgenic mice (23) and human hippocampal sections and paraffinembedded sections of 7 m in the case of human cortex.…”

“…In addition, purification of phosphorylated protein Tau from the yeast ⌬pho85 strain revealed spontaneous filament formation without the addition of anionic aggregation-inducing agents as well as the capacity of the hyperphosphorylated subfraction to drastically accelerate Tau aggregation in vitro (18). Notably, crude extracts and purified Tau preparations from humanized yeast contained higher molecular weight species, which, based on their apparent molecular weight, were tentatively identified as dimers and higher order oligomers (17,19).…”