Polyamine Uptake and Sequestering by Mammalian Cells A Two Step Two Compartment Process

In our initial studies with spermidine-C2-BODIPY (Spd-C2-BODIPY) (Fig. 1), the hypothesis that accumulation of the polyamine probe into PSVs proceeds via receptor-mediated endocytosis was initially evaluated by measuring the extent of its colocaliza-tion with fluorescent transferrin (26). The extent of such colocalization was significant, but only partial, and limited to the largest PSVs. Thus PSVs did include vesicles converging with the recycling endocytosis pathway, but other structures were clearly participating to the vesicular accumulation of the polyamine probe (26).

Our group has therefore proposed two different models to account for the vesicular accumulation of exogenous polyamine probes (Fig. 2). The first model involved the binding of the polyamine substrate to a cell surface receptor followed by the internal-ization of the resulting complex by receptor-mediated endocytosis. By analogy with the mode proposed for iron transport by DMT1 (=Slc11a2) (42), polyamines would be released from the internalized receptors as a result of endosomal acidification, and subsequently exported from PSVs to the cytosol via a vesicular membrane exporter (26,40). The second model involved a first step mediated by the translocation of polyamines into the cytoplasm via a membrane transporter or channel, followed by their rapid sequestering into PSVs via a second carrier (26,40) (Fig. 2), by analogy with the sequestering of recaptured neurotransmitters into synaptic vesicles by vesicular monoamine transporters of the Slc18 subfamily (43).

We first demonstrated that the first step in polyamine transport clearly did not rely on clathrin-dependent internalization of a hypothetical polyamine receptor because temperature-sensitive CHO cell mutants deficient in receptor-mediated endocytosis (END1) (44) displayed no defect in the uptake of Spd-C2-BODIPY (40). On the other hand, Spd^-BODIPY colocalized almost perfectly with LysoTracker Red, a probe that exclusively labels acidic subcellular compartments. Attempts to fix the internalized polyamine probe under conditions suitable for immunocytochemical studies did not succeed, and therefore it was not possible to identify the exact population of vesicles to which PSVs belong. To address this problem, we labeled instead two types of CHO mutant cell lines, LEX1 and LEX2, which bear defects in the pre-lysosome/late endosome fusion mechanism and in the maturation of multivesicular bodies, respectively (45,46). Abnormal structures accumulating in LEX1 mutants can be readily identified as prelysosome/late endosome hybrid structures in the form of perinuclear fusiform aggregates centered around microtubule organizing centers. Likewise, LEX2 mutants accumulate larger number of multivesicular bodies than parental cells. Colocalization studies demonstrated that PSVs coincide with both types of LysoTracker Red-positive, aberrant vesicles in LEX1 and LEX2 mutants, to an even greater extent than in wild-type cells (40). Therefore, one could assign PSVs to vesicles belonging to the late endocytic pathway, including multivesicular bodies, lysosomes, and late endo-somes. Interestingly, Spd-C2-BODIPY was found to accumulate to a subpopulation of the trans-Golgi network (TGN), which is also characterized by an acidic lumen (40).

A plausible explanation for the selective accumulation of the polyamine probe into acidic vesicles might have been "amine trapping." The latter phenomenon involves free

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Polyamine Cancer

Fig. 2. Two simple models initially proposed to account for the intravesicular pattern of accumulation observed for polyamine fluoroprobes. Model A, the two-step (or two-permease) model: the polyamine is first transported via a classic permease or a channel across the plasma membrane into the cytosol, and is subsequently transported inside vesicular structures (or polyamine-sequestering vesicles, PSVs) via a second transporter; sequestered polyamines could be secondarily released from these vesicles according to cellular needs. Model B, the receptor-mediated endocytosis (or single-permease) model: the polyamine binds to a cell-surface receptor, and the polyamine-receptor complex is then internalized by endocytosis. Acidification of the endosome favors dissociation of the complex, and the free polyamine exits from the endosome (e.g., using a protoncoupled symporter).

Fig. 2. Two simple models initially proposed to account for the intravesicular pattern of accumulation observed for polyamine fluoroprobes. Model A, the two-step (or two-permease) model: the polyamine is first transported via a classic permease or a channel across the plasma membrane into the cytosol, and is subsequently transported inside vesicular structures (or polyamine-sequestering vesicles, PSVs) via a second transporter; sequestered polyamines could be secondarily released from these vesicles according to cellular needs. Model B, the receptor-mediated endocytosis (or single-permease) model: the polyamine binds to a cell-surface receptor, and the polyamine-receptor complex is then internalized by endocytosis. Acidification of the endosome favors dissociation of the complex, and the free polyamine exits from the endosome (e.g., using a protoncoupled symporter).

diffusion of the uncharged form of weakly basic, amphipathic amines (e.g., LysoTracker Red) through the membrane of acidic compartments, followed by protonation of the amino group and selective retention of the cationic species inside the vesicle (47). However, as with natural polyamines, Spd-C2-BODIPY does not qualify a weakly basic compound because of the high pK values of its primary amino groups. Definitive evidence that PSV labeling by polyamine probes does not merely result from amine trapping came from the demonstration that polyamine transport-deficient CHO cells possess a normal complement of acidic vesicles but are completely resistant to labeling by Spd-C2-BODIPY (40).

Therefore, these colocalization studies identified a substantial percentage of PSVs as late endocytic and TGN vesicles. Such a specific distribution was consistent with the hypothesis that polyamines accumulate into PSVs via an antiporter-mediated mechanism that uses the outwardly directed proton gradient as a free energy source for transport, much as with the mechanism responsible for neurotransmitter sequestration into synaptic vesicles by vesicular monoamine transporters (43). Indeed, bafilomycin a potent inhibitor of the vacuolar H+-ATPase (V-ATPase), potently suppressed accumulation of the probe inside PSVs by more than 90%. However, a marked increase of diffuse cytoplasmic labeling was observed in bafilomycin-treated cells, indicating that V-ATPase inhibition still allows residual internalization of the probe. Conversely, when cells were preloaded with Spd-C2-BODIPY and then incubated with bafilomycin Aj in the absence of the probe, the fluorescent polyamine slowly emptied from PSVs over the course of more than 2 h. Initial PSV labeling at the start of chase persisted for at least 30 min, despite a complete dissipation of the proton gradient. Interestingly, the progressive decrease in intensity of PSV labeling was paralleled by a transient increase in diffuse cytoplasmic labeling (40).

It is clear from the latter studies that the pattern of vesicular accumulation of fluorescent polyamines results from their active transport into multivesicular bodies, late endosomes, lysosomes, and TGN vesicles, at the expense of the proton gradient maintained by the V-ATPase (Fig. 3). Proton-pumping activity is required not only for polyamine influx into the PSVs, but also for their vesicular sequestration, indicating the existence of an inverse pathway for efflux across the PSV membrane. Moreover, enrichment in intermediate stages of accumulation as a result of V-ATPase inhibition supports the notion that the polyamine probes accumulate or efflux from PSVs directly from or onto the cytosol.

That classic, receptor-mediated endocytosis is not required for polyamine accumulation does not rule out clathrin-independent modes of endocytosis, including caveolar endocytosis (or potocytosis) (41,48). The only experimental support for such an initial step of polyamine delivery to the cytoplasm has come from studies on spermine release from glypican-1 via a caveolin-dependent endocytic process (41). However, putrescine and spermidine do not significantly bind heparan sulfate (49), and it is thus unlikely that such a mode of spermine entry reflects a general mechanism for the polyamine transport system. Furthermore, a key role for caveolin-1 in polyamine internalization is difficult to reconcile with the known downregulation or suppression of caveolin-1 expression in many cancer types (50) and the elevation of polyamine uptake activity

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