Expandable pool

Exogenous Isbelled phenylalanine rapidly equilibrates with cytoplasmic pools in hyphae of emerged cultures of P, cycloplum. If not incorporated into protein it is accumulated in the vacuole (expandable pool). At high exogenous concentrations practically all phenylalanine needed for protein synthesis comes from the extracellular source, contrary to alkaloid synthesis which under all conditions tested recruits more than 90 % of the required phenylalanine from intracellular sources. Two pathways of alkaloid labelling can be distinguished ... 

A slowly equilibrating pool is found whose size varies rith exogenous amino acid concentration (expandable pool, cf. ref. 6). Depending on the conditions, up to 97 % of free amino acids taken up from outside may be sequestered into the expandable pool and have no direct influence on the labelling of TCA-insoluble material. 

In fungal cells (Neurospora, yeasts) the expandable pool was shown to be localized in vacuoles which can be isolated (4, 10) and contain their own specific transport system in the tonoplast membranes (10 Thus vacuoles are main storage sites of amino acids of fungal (and plant ) cells. Further, they may function as dominant regulatory agents, if uptake and release are responsive to changes in cytosolic pool sizes (5). 

The experiments in this paper will describe the intracellular distribution of externally applied labelled L-Phe between TCA-soluble, protein and alkaloid fractions. They will demonstrate that the general picture just outlined is also valid for P. cyclopium. Especially the role of cytosolic and expandable pools for labelling of secondary products v ill be discussed. 

Release of TCA-soluble radioactivity from expandable pool can be best described by the following exponential equation ... 

Rates of alkaloid labelling are very similar under pulse and chase conditions (cf. data in table). In contrast chase labelling of the protein fraction is lacking in samples pulsed vith 4 and 19 /Ug/ml L-Phe. It can be found only in discs on 50 /Ug/ml L-Phe (cf. Table, values in square). It appears that for phenylalanine released from expandable pool the opposite holds true as for phenylalanine entering cells from outsides radioactivity is preferably incorporated into alkaloids and only at rather high concentrations labelling of proteins becomes measurable. 

A third carrier system is supposed to be responsible for L-Phe accumulation into the vacuole (expandable pool, cf. (10)). It should be a low-affinity system, because significant amounts of L-Phe are only accumulated at external concentrations > 4 /Ug/ml and/or in presence of CH. Maximum capacity of this Vac... 

If protein synthesis is inhibited by CH considerable amounts of L-Phe from intracellular sources transit to the expandable pool. This leads to secondary labelling of alkaloids during pulse and labelling of proteins during chase even at lov/ concentrations of exogenous L-Phe ( <1 yug/ml). 

In the hyphal cells of P. cyclopium two pools exist for L-phenylalanine a low capacity, peripheral p6ol (the cytoplasm), and a central , expandable pool (the vacuoles) (Fig. 9). L-Phenylalanine is transported by several carrier systems through the plasma membrane and by an active transport system through the vacuole membrane. 

L-Phenylalanine in the cytoplasm serves protein and alkaloid biosynthesis. Excess L-phenyl-alanine is accumulated in the vacuoles and can be reused later in alkaloid formation but not in protein biosynthesis. Hence, with respect to alkaloid formation there are two channels for L-phenylalanine a direct, low capacity pathway via the peripheral pool (primary labeling of alkaloids) and an indirect, high capacity pathway from the expandable pool (secondary labeling of the alkaloids). The relative contributions of these two channels vary with the concentration of L-phenylalanine, the time of incubation, etc. Under all experimental conditions, however, in contrast to protein biosynthesis, about 90 % of the L-phenylalanine incorporated into the alkaloids is recruited from endogenous sources, i.e., de novo synthesis and protein degradation. 

Bulk 15 ug/ml Expandable pool of free sterol in absence of other sterol sources. Cell cone.=0.3 to 3.0 ug/mg d.w. 

---