UBC Theses and Dissertations
Potassium transport in Chlamydomonas reinhardtii Malhotra, Bhupinder K.
Several lines of evidence have indicated that K⁺ uptake is mediated by both protein carriers and channels according to external [K⁺]. Evidence derived from biophysical, biochemical and molecular studies of K⁺ uptake in higher plants, fungi and bacteria indicate that K⁺ uptake is mediated by proteinaceous carriers (active transport) and channels (passive) depending upon the external concentration of K⁺. Only limited work has been undertaken using microalgae. In Chiamydomonas reinhardtii, K⁺ influx appears to be mediated by two discrete transport systems, a high affinity transport system (HATS) at low external [K⁺] and a low affinity transport system (LATS) at high external [K⁺]. These two transport systems were further characterized by employing various metabolic inhibitors, a K⁺ channel blocker and a sulfhydryl reagent. Effects of light and dark on K⁺ transport were also studied. To calculate Δμk⁺ across the plasma membrane, electrical potential difference (Δψ) was measured using TPP⁺, and cytoplasmic [K⁺] was obtained from compartmental analysis. According to this value of Δμk⁺, K⁺ enters the cell against its electrochemical potential gradient at low [K⁺]₀ (<0.2 mM) and is probably moving down the electrochemical gradient at high [K⁺]₀ (>1 mM). In isolated chloroplasts, [K⁺] was found to be 165±21 mM. This value was close to that estimated by compartmental analysis (223±31.4 mM). Compartmental analysis of cells using ⁸⁶Rb⁺ and ⁴²K⁺ as the tracers also showed the presence of three cellular compartments and their half-lives. Three compartments detected in Chiamydomonas probably correspond to the cell wall, cytoplasm and chioroplast, respectively. Comparisons of compartmental analysis of wild type and trkl cells showed that compartment III (putatively identified as the chioroplast) may serve as a reservoir for K⁺. It is a common observation that plants show increased ion uptake when deprived of nutrients. Chiamydomonas showed increased uptake of K⁺(⁴²K⁺or ⁸⁶Rb⁺) when K⁺ supply decreased in the external medium. Inhibitors of protein synthesis were used to study the role of de novo protein synthesis in the development of increased K influx associated with K⁺ deprivation. There was no significant increase in K⁺ uptake before 3 h of K⁺ deprivation. K⁺ influx increased significantly after 3 h and peaked at about 18 h of K⁺ deprivation. In parallel experiments, expression of membrane polypeptides was studied. Some polypeptides in response to K⁺ deprivation were expressed in the microsomal fraction. When cells were deprived of K⁺ for 4 h, a 22 kD polypeptide showed increased expression. Another polypeptide of Mr 51 kD was synthesised as a response to long term K⁺ deprivation. When comparisons were made between microsomal fractions from WT and trkl cells, a 21 kD polypeptide was found to be absent from trkl microsomes.
Item Citations and Data