We have shown previously that cells expressing a GDP-preferring Rab9 mutant protein (Rab9 S21N) display defects in MPR transport from endosomes to the Golgi complex (Riederer et al., 1994
). To investigate further the importance of Rab9 for the function and morphology of late endosomes, we used siRNA to deplete Rab9 from cultured cells. Immunoblot analysis showed that Rab9 protein was decreased >90% upon siRNA treatment (). The depletion seemed to be specific for Rab9 as the steady-state level of the late endosomal Rab7 protein was unchanged (). Loss of Rab9 did not alter the steady-state level of TIP47. This may not be surprising because the majority of TIP47 is cytosolic and that pool of TIP47 does not contain bound Rab9 (Diaz and Pfeffer, 1998
). Similarly, the level of the late endosome/lysosomal marker LAMP1 increased only slightly upon Rab9 depletion (58%, ; see below). However, loss of Rab9 led to a more significant increase in CI-MPR protein levels ().
Figure 1. Rab9 depletion destabilizes CI-MPRs and induces their expression. (A) Immunoblot analysis of HeLa cell lysates (equal protein amounts) treated for 72 h with luciferase control siRNA (-) or Rab9 siRNA (+) by using the indicated antibodies. (B) HeLa cells, (more ...)
The increase in CI-MPR levels was not due to a block in degradation: the MPR half-life decreased from 34 to 25 h, consistent with a small increase in MPR missorting to the lysosome (). Furthermore, careful examination of the levels of CI-MPRs detected at time 0 of this pulse-chase, protein biosynthesis experiment revealed a roughly 30% increase in CI-MPR protein biosynthesis rate. Over the 25 h half-life of the CI-MPR in cells lacking Rab9, we would therefore expect an increased accumulation of CI-MPRs that would be counterbalanced by the increase in degradation to yield a roughly twofold increase at steady state. Induction of MPR expression was seen previously in cells expressing Rab9 S21N (Riederer et al., 1994
); cells expressing higher levels of MPRs can deliver newly synthesized lysosomal enzymes to the lysosome in the absence of MPR recycling.
The functionality of late endosomes depleted of Rab9 was analyzed by measuring the rate of EGF-stimulated EGF receptor degradation. This process is expected to be independent of Rab9 protein because it involves transport of EGF receptors to the lysosome. As shown in , the rate of EGF receptor degradation was unchanged in cells depleted of Rab9: EGF receptor half-life was close to 37 min in both control and depleted cells. Thus, Rab9 depletion does not seem to perturb endosome-to-lysosome transport.
We next examined the morphology of late endosomes in cells lacking Rab9 (). In the absence of Rab9, Rab7 displayed a more restricted, perinuclear localization with an apparent reduction in the number of larger, peripheral vesicular structures compared with control cells (, bottom left and right). Similarly, LAMP1 staining revealed a reduction in larger, peripheral spherical structures accompanied by a much tighter perinuclear localization in Rab9-depleted cells (, left and right). The tighter perinuclear staining in the absence of Rab9 could be explained if Rab9 normally recruits a yet-to-be-identified motor protein involved in late endosome distribution. The LAMP1 distribution seemed to be more significantly concentrated than that of Rab7; this could be due to the presence of a portion of Rab7 on earlier compartments that do not contain Rab9. Alternatively, loss of Rab9 may lead to the release of a motor protein from late endosomes that is now available for clustering of LAMP1-positive lysosomes.
Figure 2. Late endosome morphology is altered in Rab9 depleted cells. (A) Immunofluorescence microscopy of HeLa cells transfected with control siRNA (left) or Rab9 siRNA (right) and double labeled with monoclonal anti-Rab9 antibody (top) or polyclonal anti-Rab7 (more ...)
A possible reduction in the size of late endosomes in Rab9-depleted cells was explored further using whole-mount electron microscopy (). Stoorvogel and coworkers have devised a method to grow cells directly on EM grids and to label endosomes selectively by endocytosis of HRP or conjugates thereof (Stoorvogel et al
., 1996). Cells were incubated with HRP for 60 min at 37°C followed by a 30-min chase to fill late endosomes (and not lysosomes). Samples were then fixed, permeabilized, and visualized in whole mount to view all late endosomes in both control and Rab9-depleted cells. Late endosomes look generally like perinuclear structures with vesicular and tubular elements (Stoorvogel et al
., 1996; Kleijmeer et al. 2001
; ). Notably, in Rab9-depleted cells, late endosome diameters decreased by 45% (p < 0.01) from an average of 0.76 to 0.42 μm (). The cross-sectional area of individual late endosomes was decreased correspondingly ~70% (p < 0.01) from an average area of 0.37 to 0.11 μm2
(). Thus, Rab9 knockdown reduces the size and volume of individual late endosomes.
Figure 3. Rab9 depletion decreases late endosome size. (A) HeLa cells transfected with control siRNA (left) or Rab9 siRNA (right) were labeled by fluid phase endocytosis of HRP for 1 h at 37°C followed by a 30-min chase. Cells were then processed for whole-mount (more ...)
We used conventional, thin-section electron microscopy to examine the structure of late endocytic compartments in HeLa cells treated with or without Rab9 siRNA (). Three types of late endosomes/lysosomes were readily identified in HeLa cells, which we refer to as dense tubular, multilamellar, and multivesicular structures (). Multivesicular endosomes contained one or more internal vesicles, multilamellar endosomes/lysosomes contained concentric layers of membranes or membrane whorls, and dense tubular endosomes/lysosomes contained a dense network of internal membranes and may represent the MPR-enriched late endosomes described by Griffiths et al
) (, left). These latter structures were either present as single entities or were found in larger, membrane-bound compartments in combination with one or more of the three structural types. Immunogold labeling further confirmed that each of these structural classes included late endosomes because both Rab7 (large gold) and Rab9 (small gold) could be detected on their surfaces (). Although not all Rab7 and Rab9 colocalize on individual late endosomes (, left), examples of structures containing both proteins were readily detected by immunoelectron microscopy (). It is important to note that most anti-Rab9 staining was seen on the multivesicular endosome class, which includes both early and late endosomes subtypes.
Figure 4. Late endosome subclasses in HeLa cells. Electron micrographic examples of dense tubular (left column), multilamellar (middle column), and multivesicular (right column) endosomes. Bar, 200 nm; magnification was the same for all panels. (B) HeLa cells processed (more ...)
Late endosomes are concentrated close to the microtubule organizing center. This area was identified in sections of control and Rab9-depleted HeLa cells and the number of each late endosome structural class was quantified (). Multivesicular late endosome numbers remained unchanged in control and Rab9 depleted cells, with approximately four structures per 5 μm2
. This is perhaps not surprising because multivesicular endosomes are generated from early endosomes through the sequential action of the endosomal complex required for transport (ESCRT) complexes (Katzmann et al., 2001
; Babst et al
). Thus, most of these structures are likely to represent predominantly, endosomes located earlier in the endocytic pathway than those that contain Rab9.
Dense tubular and multilamellar late endosomes/lysosomes were less common than multivesicular endosomes in a given section, averaging only one to two structures per 5 μm2 in the perinuclear region of control cells (). Surprisingly, we found a clear reduction in the number of both of these structural classes in Rab9-depleted cells, with less than one structure identified per 5 μm2 (). Therefore, Rab9 depletion influences the maintenance or generation of the dense tubular and especially, multilamellar structures. In addition to these subsets of endosome/lysosome compartments, essentially all kinetically defined late endosomes labeled in were reduced in size. It is important to note that most of the endosomes labeled in are likely not multilamellar or dense tubular structures, despite their decrease in size upon Rab9 depletion. This conclusion stems from our finding that most late endosomal Rab9 is detected on multivesicular endosomes by immunoelectron microscopy.
How might Rab9 depletion decrease endosome size? In control-treated HeLa cells, prenylated Rab9 may be accom-modated in membranes by an increase in late endosome membrane surface. Alternatively, Rab9 may play a role in the generation and/or maintenance of a larger subclass of late endosomes/lysosomes; in the absence of Rab9 these structures might fail to form or have a shorter lifetime. Because Rab9 is essential for the delivery of newly synthesized lysosomal enzymes to lysosomes, perhaps the loss of dense tubular and multilamellar structures simply reflects a primary defect in lysosome formation.
Biochemical Characterization of Dense Lysosomes
Electron micrographic analysis revealed a decrease in late endosome size in cells depleted of Rab9 protein (). In addition, we noted a decrease in the number of dense tubular and especially multilamellar endocytic structures (). Although most Rab9 was normally present on multivesicular late endosomes, it was important to evaluate the overall status of the lysosome compartment in Rab9-depleted cells because it was possible that the multilamellar structures were lysosomal in origin. Also, as seen previously (Riederer et al., 1994
), lysosomal enzyme levels were upregulated to compensate for the loss of Rab9 protein: LAMP1 protein and hexosaminidase activity were increased by 58 and 56%, respectively, in Rab9-depleted cells (; our unpublished data). Thus, we used a procedure devised by Kornfeld and colleagues for rapid separation of dense lysosomes, which are collected in the lower 30% (fraction 3) of a Percoll gradient (Rohrer et al., 1995
As expected, the bulk of the lysosomal LAMP1 protein was recovered in fraction 3 (, bottom). In addition, despite slight increases in lysosomal enzymes, the relative amounts of protein (, top), phospholipid (middle), and LAMP1 protein (bottom) were not significantly different when Rab9-depleted and control cells were compared. Therefore, lysosomal enzyme activity per mole phospholipid or protein did not change relative to other membrane-bound compartments as determined by this global biochemical analysis.
Figure 5. Percoll density gradient analysis of Rab9-depleted cell membranes. Control and Rab9-depleted cells were harvested and fractionated as described by Rohrer et al. (1995 ); the gradients were divided into three fractions and analyzed for protein (top), phospholipid (more ...)
Although late endosomes decreased in size (), the buoyant densities of markers that are in part present in late endosomes (hexosaminidase [our unpublished data] and LAMP1) were generally unchanged. In summary, these fractionation experiments lead us to conclude that the decrease in multilamellar structures () does not significantly influence the overall distribution of enzymes among late endosomes and lysosomes.
Rab9 Influences Trafficking of Other Late Endosome Markers
As shown in , the loss of Rab9 protein led to an increase in total cellular CI-MPR coupled with an enhancement in the rate of this protein's degradation. In addition, we noted that cells contained higher levels of surface MPRs and early endosome MPRs. This was easily detected in experiments in which cells were incubated with anti-MPR antibodies for short periods of time (15 min; ). Under these conditions, Rab9-depleted cells internalized significantly more antibody than control cells (, top). In addition, these cells internalized increased amounts of LAMP1 antibody (, bottom). Although some of the increase of cell surface MPRs may be due to an increase in their biosynthesis, LAMP1 protein levels were only increased by 58% upon Rab9 depletion (). Thus, it seems that Rab9 contributes to the retention of a fraction of MPRs and LAMP1 within late endosomes, either via microdomain incorporation or by regulation of endosome egress. In the absence of Rab9 protein, endosome-to-cell surface transport seems to be enhanced. The presence of LAMP1 on the surface is consistent with an essential role for Rab9 in early events of lysosome formation.
Figure 6. Loss of Rab9 increases cell surface CI-MPR and LAMP1. HeLa cells transfected with buffer alone (left) or Rab9 siRNA (right) were incubated with anti-CI-MPR antibody (top) or anti-LAMP1 antibody (bottom) for 15 min before extensive washing, fixing, and (more ...)
Rab9 Stability Requires Effector Interaction
As described above, endosome-associated Rab9 recruits its cytosolic effector protein, TIP47, which binds to MPR cytoplasmic domains (Diaz and Pfeffer, 1998
; Carroll et al., 2001
). TIP47 binding is thought to enrich MPRs within a Rab9 microdomain and thereby facilitate MPR collection into nascent transport vesicles. We investigated TIP47's role in stabilizing this microdomain by using siRNA to deplete TIP47 from cultured cells. Immunoblot and immunofluorescence analysis showed that TIP47 levels could be decreased up to 80% by siRNA treatment (). In the presence of TIP47 siRNA, but not a control lamin A/C siRNA, TIP47 protein levels decreased with time of treatment (our unpublished data). Steady-state levels of Rab9, Rab7, LAMP1 protein (), and CI-MPR (our unpublished data) did not change substantively upon depletion of TIP47. Moreover, immunofluorescence microscopy detected Rab9 on what seemed to be normal, perinuclear, late endosomal structures (our unpublished data).
Figure 7. TIP47 is required for Rab9 and CI-MPR stability. (A) Immunoblot analysis of equal amounts of protein from HeLa cell lysates transfected with control siRNA (-) or TIP47 siRNA (+) were analyzed using the indicated antibodies. (B) Anti-TIP47 antibody indirect (more ...)
The apparent minimal changes in the steady-state levels and localizations of MPRs and Rab9 proteins masked significant changes in protein stability as determined by pulse chase labeling of the proteins and examination of their turnover (). In previous work, antisense depletion to reduce TIP47 protein levels by ~50% led to MPR missorting to the lysosome and it reduced the CI-MPR half-life by twofold (Diaz and Pfeffer, 1998
). Thus, as expected, TIP47 siRNA decreased the stability of CI-MPR 1.9-fold, reducing the protein half-life from 26 to 14 h (). The change in turnover was not due to a general increase in protein turnover because the low-density lipoprotein receptor lifetime was unchanged (). Unexpectedly, the half-life of Rab9 protein decreased from 32 to 8 h, a fourfold decrease in stability due to loss of TIP47. The effect was specific for Rab9 because the half-life of Rab7 protein was completely unchanged ().
The apparent minimal change in the steady-state level of Rab9 protein, coupled with a greatly increased rate of degradation, suggests that Rab9 gene expression is induced upon TIP47 depletion. How TIP47 depletion triggers Rab9 transcription is unknown. As was observed for Rab9 depletion (), EGF-stimulated EGF receptor degradation was unchanged in TIP47-depleted cells, suggesting that overall late endosome function was retained (our unpublished data).
Why should Rab9 protein be much more rapidly degraded in the absence of TIP47? Investigators in this research area generally think of prenylated Rabs as either existing in the outer leaflet of an organelle membrane or bound to the carrier protein GDP dissociation inhibitor (GDI) in the cytosol. Prenylated Rabs are considered independent entities, despite their ability to interact with a variety of important effector proteins (Zerial and McBride, 2001
). Yet, when TIP47 was depleted from cells, Rab9 protein was rapidly degraded, either via proteosomal degradation or delivery to lysosomes. Thus, we cannot think of Rabs as truly independent constituents: their interactions with effectors can influence greatly, their cellular fates. In summary, TIP47 is essential for both Rab9 stabilization and MPR recycling. Together, these data support a model in which TIP47 interacts with MPRs and Rab9 protein in living cells. Moreover, these data highlight the independent regulation of Rab9 and Rab7 endosomal domains because Rab7 protein was not destabilized upon loss of Rab9 or TIP47 proteins.
Stabilization of the Rab9 Microdomain in Living Cells
Prenylated Rab proteins are delivered to membranes by GDI and are thought to be in equilibrium between membranes and a cytosolic, GDI-bound form. We have shown that in the absence of TIP47, Rab9 protein is significantly destabilized. One possibility is that Rab9 membrane association is normally favored by TIP47 binding, relative to GDI-mediated, membrane release. If true, TIP47 depletion might increase the amount of cytosolic Rab9. As shown in , we detected a twofold increase in the level of cytosolic Rab9 upon TIP47 depletion. Although ~10% of total Rab9 was found bound to GDI in control cell cytosol, depletion of TIP47 increased that value to 20% (). We cannot completely exclude the possibility that this increase reflects an increase in newly synthesized Rab9 protein en route to endosomes. Newly synthesized Rab proteins are believed to be delivered to membranes by the Rab escort protein (Alexandrov et al., 1994
), but GDI also may play a role (Sanford et al., 1995
). Nevertheless, the absence of TIP47 seems to shift the equilibrium between membrane-associated and cytosolic Rab9 protein.
Figure 8. Rab9 binds TIP47 with high affinity and shifts to the cytosol upon TIP47 depletion. (A) Equal amounts of crude cytosol and membrane fractions from control and TIP47 siRNA-transfected HeLa cells were analyzed by immunoblotting with anti-Rab9 antibodies. (more ...)
Given the importance of TIP47 for Rab9 stabilization on endosomes, we determined the affinity of interaction between these binding partners. For this purpose we made use of the fact that the presence of TIP47 in Rab9 nucleotide binding assays leads to a higher level of overall nucleotide binding (Hanna et al., 2002
). This method could thus be used as an indirect measure of physical interaction. An advantage of this approach is that it detects only active Rab9 protein binding to TIP47, and it uses binding of radiolabeled nucleotide to determine quantitatively, the amount of Rab9-bound TIP47 protein.
As shown in , the affinity of TIP47 for Rab9 bound to GTPγS was strong (Kd
= 95 nM; filled circles). The affinity of TIP47 for Rab9 bound to GDP was lower (Kd
= 160 nM; open circles). Thus, the interaction between TIP47 and Rab9 is of high affinity—almost 10-fold higher than the affinity of TIP47 for the cytoplasmic domain of the MPR (Kd
= 1 μM; Krise et al., 2000
). The high affinity of TIP47 for Rab9 is consistent with the importance of this interaction in stabilizing Rab9 protein and supports a model in which Rab9 recruits TIP47 onto late endosomes.