Cellular injury caused by gentamicin is most pronounced in kidney proximal tubule cells, which accumulate the highest concentrations of this antibiotic. Following unimpaired filtration of gentamicin across the glomerulus, cellular uptake involves both fluid-phase and receptor-mediated, megalin-associated endocytosis (21
). The majority of internalized gentamicin is delivered to the lysosome. The mechanism by which gentamicin produces cell injury is still unresolved, though recent studies from our laboratory support the importance of retrograde movement of gentamicin through the endosomal system. Utilizing TR-gentamicin in proximal tubule cells, we showed that 5 to 10% of internalized gentamicin is initially trafficked from endosomes to the Golgi (29
). Further analysis revealed gentamicin delivery to the endoplasmic reticulum, with subsequent cytosolic release and subcellular compartment association, including the nucleus and mitochondria (32
). Furthermore, we observed a reduction in mitochondrial potential in gentamicin-treated cells which was at times consistent with cytosolic delivery of gentamicin, suggesting one possible mechanism of toxicity. The movement of gentamicin through multiple membrane-bound organelles and its eventual cytosolic delivery would potentially enable this polycationic antibiotic to encounter and disrupt multiple biochemical processes occurring in both membrane and cytosolic locations. The mechanism(s) by which gentamicin undergoes retrograde trafficking to the ER may involve pathways utilized by other cellular toxins (20
). Therefore, investigating the trafficking and interactions of gentamicin in a eukaryotic model system that is more amenable to genetic, biochemical, and molecular dissection could lead to a better understanding of the pathophysiology of gentamicin and other toxins.
The recent screening of a yeast deletion library for antibiotic-sensitive strains by Blackburn and Avery (3
) uncovered many gentamicin-sensitive strains possessing mutations in proteins involved in intracellular trafficking pathways. These observations provided findings for the eukaryotic model system that were consistent with our mammalian studies (29
). Therefore, we set out to further characterize the specific pathways and protein complexes, which if deleted, would lead to increased gentamicin sensitivity. We also utilized TR-gentamicin as a tool to begin characterizing its transport in yeast and evaluating differences between the gentamicin-sensitive strains as a tool to further understand cellular toxicity.
We did note many similarities and some differences between our findings and those of Blackburn and Avery (3
). These differences most likely resulted from differences in the sensitivities of our screens. Their screen was performed under a single condition (YPD with 256 μg/ml gentamicin and growth evaluated after 2 days at 30°C). While false positives were unlikely to be overlooked due to the careful analysis of mutants after the preliminary screen, the uneven growth rate of the strains within the mutant collection as well as the uneven number of cells present in each initial culture could have caused difficulties in determining antibiotic sensitivities with a fraction of the mutants.
Our results, as shown in Fig. , suggest that the deletion of a protein from any of several intracellular vesicular pathways and or complexes resulted in sensitivity to gentamicin. One such complex that appeared critical for tolerating gentamicin is the NAC containing Zuo1 (13
). Zuo1 is a ribosome-associated J protein and a partner of Ssb1 and Ssb2 of Hsp70 and the Hsp70-related protein Ssz1. Together, they act as a molecular chaperone to facilitate protein folding of nascent proteins. Their absence clearly caused gentamicin sensitivity, and a recent study suggests the mechanism may be due to altered plasma membrane function that results in ion transport changes (14
). Kim and Craig also suggested this chaperone complex may participate in the folding of WD40 proteins, many of which are involved in the secretory pathway (15
). Their studies emphasized the importance of understanding both a protein's interaction(s) and the cellular pathway(s) in which it participates.
FIG. 7. Diagram showing the location of several complexes (HOPS, GARP, and NAC) and individual genes whose absence results in gentamicin-hypersensitive strains. The delivery of gentamicin to the Golgi, ER, and vacuole and cytoplasmic interaction with ribosomal-associated (more ...)
Two other protein complexes, HOPS and GARP, have multiple members whose absence resulted in significant gentamicin sensitivity. Blackburn and Avery identified four members of the HOPS complex (Pep3, Pep5, Vps33, and Vps16) as affecting gentamicin resistance (3
). In our growth assay, cells with VPS16
mutations were gentamicin sensitive while cells lacking the other HOPS components were minimally affected. We also examined cells with two other HOPS-member mutations (Vam6 and Vps41) and found that the loss of Vps41 resulted in an approximately 50% growth inhibition in the presence of gentamicin, while the loss of Vam6 caused little growth retardation. Interestingly, the loss of Vps8, which has an association with Pep3, Pep5, Vps16, and Vps33 based on affinity precipitation, resulted in a growth inhibition of greater than 50%. The HOPS complex plays an important role in vacuole fusion events (40
). The variable effects we observed in the HOPS deletion mutants suggest either that the vacuole fusion event is not important in inducing gentamicin sensitivity or that some HOPS members participate in other pathways with a greater effect on gentamicin sensitivity. In support of the latter, Vps41 has been shown to be involved in both vesicle budding and fusion during vacuole biogenesis and Vps16, whose loss results in one of the most gentamicin-sensitive strains, was shown to have more than a 50% reduction in Dcp1-dependent mRNA decapping activity (23
). Reduction in decapping activity would remove a key signal needed to increase translation, which is potentially important for adaptation following gentamicin exposure.
Vps54 (Luv1) was identified by Blackburn and Avery as important for gentamicin resistance though they did not discuss its role as a component of the GARP complex (3
). This complex contains at least four proteins (Vps51, Vps52, Vps53, and Vps54) and has an associated Rab, Ypt6, in addition to interacting with various t- and v-SNAREs, and the SM protein, Vps45 (39
). This complex has a critical role in the docking and fusion of endosome-derived vesicles with the trans
-Golgi network. The absence of any of the GARP members resulted in significant and similar gentamicin sensitivities. This is not surprising for Vps52, Vps53, or Vps54 since each of these proteins is necessary for the stability of the remaining components of the complex, and the absence of any one of them results in the rapid degradation of the other two (10
). In contrast, loss of Vps51 does not eliminate the other GARP members (26
). Consequently, the similar gentamicin sensitivities we observed in cells with Vps51 mutations or the other GARP members support our conclusion that the loss of the GARP complex is a key contributor to inducing gentamicin toxicity. In further support of this, the deletion of YPT6
, or VPS45
resulted in significant gentamicin sensitivity. The loss of Vps45 results in down-regulation of Tlg2 but not Tlg1, and the truncation of Tlg2 inhibits the association of Vps45 with the t-SNAREs (7
). The increased gentamicin sensitivity caused by loss of Vps45 compared to loss of Tlg2 suggests that the loss of Vps45 affects another pathway in addition to GARP. Recent studies have shown the importance of this SM protein in both yeast and mammalian cells. There are only four SM proteins in yeasts and seven in mammals, while many more SNAREs exist (39
). The SM proteins are essential for intracellular membrane fusion events and appear to work closely with specific SNAREs to provide specificity in the membrane fusion process (11
). Vps45 and Tlg2 have also been shown to have a role in the constitutive Cvt pathway (1
). Future analysis of strains containing specific point mutations of the gentamicin-sensitive deletion proteins and double mutants will help to define what pathways and interactions, when absent, result in toxicity.
The final two strains that exhibited significant gentamicin toxicity had SAC1
is present on the opposite DNA strand but overlaps YDR455c
). We found similar results when analyzing either YDR455c
mutants. Nhx1 is one of the oldest members of the Na+
exchanger family (5
). Members of this family have important roles in multiple functions including salt tolerance, transepithelial Na+
transport, vesicle trafficking, and vesicle biogenesis. Several studies have shown the importance of Nhx1 in endosomal trafficking, where it appears to regulate the pH of endosomes (2
mutants missort CPY and have a general defect in vesicle trafficking out of the endosome that appears to be a pH-dependent event. SAC1
codes for a lipid phosphatase that has been localized to ER and Golgi membranes (12
). Its enzymatic activity acts on phosphatidylinositol 4-phosphate [PtdIns(4)P]. SAC1
mutants accumulate PtdIns(4)P at ER and vacuolar membranes, which in turn results in altered late endocytic and vacuolar trafficking (37
). A common attribute of many of the gentamicin-sensitive mutants is altered trafficking involving endosomes. This information, when taken together, enables better predictions to be made concerning how and why specific gene deletions result in increased gentamicin toxicity.
Our previous studies in mammalian cells documented the utility of TR-gentamicin as a probe to monitor the intracellular location of gentamicin. In the present studies, the level of TR-gentamicin uptake correlated qualitatively with growth inhibition and LY uptake (Fig. ). To quantitatively address uptake, we utilized OG-gentamicin and flow analysis (Fig. ). While there is a clear positive correlation between gentamicin uptake and dead/injured cells, additional studies are needed to establish for each strain whether localization or uptake is the most critical parameter. Delivery of gentamicin to a specific cellular location may be critical for inducing cell toxicity. Localization of TR-gentamicin clearly showed that delivery to LY staining vacuole structures was the predominant pathway. This is consistent with mammalian studies in which the majority of intracellular gentamicin was localized to the lysosome. A critical question is whether gentamicin is trafficked to other locations and/or whether it causes missorting in the trafficking pathways. The present studies support the importance of the GARP, HOPS, and NAC protein complexes whose absences resulted in gentamicin-sensitive strains. Visual analysis of these strains using TR-gentamicin suggested localization to smaller punctate structures, possibly endosomes, and fragmented vacuole pieces for the GARP mutants. Distinguishing fragmented vacuole localization of TR-gentamicin from endosomal or cytosolic locations will require further investigation. However, the known effect of gentamicin on membrane fusion and documented cytosolic release in mammalian cells suggests that cytosolic release is a likely mechanism by which gentamicin induces toxicity in both yeast and mammalian cells.