Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Abstract

b Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K ؉ ] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K ؉ and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ϳ500:1, viz., 10-to 50-fold larger than that in K ؉ -and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K ؉ stress by means of progressively increasing transporter affinity for extracellular K ؉ . The underlying observations are (iv) that K ؉ influx via Trk1p remains nearly constant at ϳ9 mM/h when extracellular K ؉ is progressively depleted below 0.05 mM and (v) that K ؉ influx via Hak1p remains at ϳ3 mM/h when extracellular K ؉ is depleted below 0.1 mM.

The Role of Soil Fungi in K+ Plant Nutrition

“…On the other hand, TRK1 was not found to be transcriptionally regulated, while HAK1 was strongly induced in response to very low K + concentrations and was down-regulated by the presence of K + . Similar behaviors were shown in the homologous systems of Neurospora [53,61].…”

Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

“…Also lacking are publicly-available whole-transcriptome data sets such as has been used for other organisms when selecting reference genes. Therefore, potential reference genes were selected based on the following features: previous transcriptomic-based studies from N. crassa ( acl ) [25] or other fungi ( adk , asl ) [23] in which the expression of these genes remained unchanged; via selection from the Broad Institute N. crassa database ( http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html ) with genes for which no expression data existed ( tbp ), or from previous studies of genes whose results pertaining to their function and regulation indicated the potential for use as a reference gene ( vma1 , vma2 , vma3 , trk) [26] , [27] Additionally, N. crassa genes employed as reference genes in previous studies were also evaluated here ( act , btl , l6 , frh ) [28] , albeit with newly-designed primers for use in the btl and frh assays.…”

Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains

“…2, calibrating sample band intensities by means of HA-tagged plasma membrane ATPase (ScPma1-HA; enzyme provided by A. B. Mason) as described previously (34). Samples were assayed for total protein content using the Lowry method (35), before being electrophoresed on precast polyacrylamide gradient gels (4 -15% TGX gel 456 -1086; Bio-Rad), and then stained for 1 h with primary anti-HA monoclonal antibody (MMS-101P, Covance Corp., Berkeley, CA) and for a 2nd h with antimouse antibody coupled to horseradish peroxidase (HRP; 4021, Promega Corp., Madison, WI).…”

“…We determined the amount of FEX protein in yeast plasma membranes using quantitative Western blotting with HA-tagged versions of Fex1p and Fex2p and a calibration standard (34). When expressed separately, Fex1p and Fex2p are present in yeast membranes at the same level, 1.7 or 1.8 fmol/g of membrane protein, respectively.…”