Reduced endosomal microautophagy activity in aging associates with enhanced exocyst‐mediated protein secretion

Previous data showed that LE/MVBs from old animals accumulate undegraded oxidized proteins (Cannizzo et al., 2012), suggesting a possible age‐related decline in eMI function. To investigate this possibility, we isolated LE/MVBs from young (4–6 m) and old (22–24 m) mouse livers and reconstituted the different steps of eMI in vitro using a previously developed assay (Krause & Cuervo, 2021; Sahu et al., 2011) (Figure 1a; comparable purity of fractions isolated from young and old mice was verified by immunoblot for LE/MVBs markers and enzymatic assays for the endolysosomal enzyme β‐hexosaminidase [Figure S1a–c]). LE/MVBs were incubated with two KFERQ‐motif containing proteins (Tau and α‐synuclein), previously shown to undergo degradation by selective eMI (Caballero et al., 2018; Krause et al., 2022) or with Cyclophilin A, which lacks a KFERQ motif and can be internalized and degraded in LE/MVBs through non‐selective eMI (Sahu et al., 2011) (Figure 1b). Incubation in the presence or absence of protease inhibitors allows for the differentiation of substrate binding (substrate recovered in LE/MVBs not treated with protease inhibitors) and substrate internalization/degradation (calculated as the difference between the substrate recovered in LE/MVBs pre‐treated with protease inhibitors and those untreated) (Figure 1a). We found reduced binding for the three proteins in LE/MVBs from old mice (Figure 1b,c), and significantly reduced internalization/degradation for both selective substrates, Tau and α‐synuclein, that was not observed for Cyclophilin A (Figure 1b,d), suggesting that the functional decrease with age preferentially affects KFERQ‐selective eMI.

Defective eMI with age could be a consequence of changes in activity per LE/MVB or in the fraction of cellular LE/MVBs that can perform eMI. To differentiate between these possibilities, we incubated LE/MVBs with the eMI substrate Tau and performed immunofluorescence of the glass‐spotted samples for Tau and the endolysosomal marker LAMP‐1 to measure the percentage of LE/MVBs competent for eMI (LAMP‐1⁺/Tau⁺) (Figure 1e). We observed no difference between the two ages in the percentage of LE/MVBs (LAMP‐1⁺ vesicles) that colocalized with the eMI substrate Tau (Figure 1f), suggesting that the fraction of LE/MVBs capable of performing eMI is preserved with age and that it is the absolute eMI activity per LE/MVB that decreases in the old group.

To determine if the reduction in eMI activity with age is observed in vivo and to explore possible changes in substrate targeting to LE/MVBs with age (a step not reconstituted in the in vitro system), we injected young and old mice with saline or the endolysosomal protease inhibitor leupeptin to prevent degradation in LE/MVBs (Figure 1g,h; efficacy of leupeptin injection was confirmed in total homogenate with proteins known to undergo lysosomal degradation [Figure S1d]). Immunoblot for three KFERQ‐bearing proteins (GAPDH, Aldolase and LRRK2) did not reveal reduction with age in the levels of endogenous eMI substrates associated with LE/MVBs (Figure 1i), suggesting that Hsc70‐mediated targeting to these organelles was comparable in young and old mice. However, once again, we found clear trends of reduced internalization/degradation for two of the selective endogenous substrates (Aldolase and LRRK2) (Figure 1j), which match their higher steady‐state levels in this compartment (Figure 1i) (likely bound to the membrane but not efficiently internalized/degraded). This decrease in eMI was not observed for one of the other endogenous substrates (GAPDH), suggesting possible substrate‐specific differences in their ability to undergo eMI with age and likely competition for LE/MVB internalization/degradation based on the cellular abundance of these proteins.

We next used a modified in vitro assay to analyze substrate internalization separately from degradation by measuring the proteolysis of a pool of radiolabeled proteins incubated with isolated LE/MVBs with intact membranes or upon disruption of their membranes with detergent to allow direct access of the LE/MVB enzymes to the radiolabeled substrates, and thus, eliminate the internalization step. We found a marked reduction in the degradation of radiolabeled proteins in both cases (Figure 1k), suggesting that at least part of the age‐related reduction in eMI is due to less efficient proteolysis by luminal enzymes.

Despite lower proteolytic activity, immunoblot of isolated LE/MVBs from old mice did not reveal major changes with age in levels of the luminal enzymes GBA and Cathepsin D (Figure S1a,b). We next considered the possibility of defective acidification. However, measurement of the pH of isolated LE/MVBs using the radiometric pH probe LysoSensor Yellow/Blue DND‐160 revealed higher acidification in old mice LE/MVBs (pH shift from 5.75 to 5.36) (Figure 1l). Direct measurement of the activity of other pH‐dependent enzymes in LE/MVBs such as β‐hexosaminidase, which hydrolyzes amino sugars, also confirmed higher activity in LE/MVBs from old mice (Figure S1c), further supporting their proper acidification.

Overall, these findings unveil a decline in selective eMI with age mostly due to defective degradation of internalized cargo. Reduced substrate degradation does not seem to be a consequence of overall changes in LE/MVB properties such as acidification but appears to be instead limited to defective protein break down.

We next sought to understand the molecular defects behind the functional decline in eMI by analyzing possible changes in the levels of eMI‐related components with age. Immunoblot analysis revealed that total cellular levels (Figure S2a) and LE/MVB levels (Figure 2a) of eMI components were comparable in both ages, with the exception of Hsc70, which is increased 2.5‐folds in LE/MVBs from old mice. The elevated levels of Hsc70 in old LE/MVBs was driven by an additional higher molecular weight band immunoreactive for Hsc70 only in LE/MVBs from old mice (Figure 2a). Treatment of LE/MVBs from old mice with different endoglycosidases to remove sugar moieties led to a discrete decrease in the molecular weight of the upshifted Hsc70 band after treatment with Endoglycosidase H (Endo H), which deglycosylates mannose glycoproteins (Figure S2b; LAMP‐1 is shown as positive control of glycosylated protein); whereas incubation with PNGase F, which removes all types of asparagine‐linked (N‐linked) glycosylation and advanced glycosylation end‐products from glycation, eliminated the high molecular weight form of Hsc70 (Figure 2b; LAMP‐2A is shown as positive control of glycosylated protein), consistent with an age‐related sugar modification as previously detected for other proteins in LE/MVBs from old organisms (Cannizzo et al., 2012). While this modification is detectable in liver homogenates from old mice (27% of the total cellular Hsc70), it is highly enriched in LE/MVBs where it represents 53% of LE/MVB‐associated Hsc70 (Figure 2c,d). It is unlikely that the glycosylated form of hsc70 in LE/MVBs is a cytosolic contaminant, since only after long exposure a small fraction of cytosolic hsc70 (<10%) displayed the molecular weight shift (Figure S2c). Mass spectrometry analysis identified that Hsc70 in LE/MVBs from old mice had sugar moieties in five asparagine residues not present or barely detectable in Hsc70 from young LE/MVBs (Figure 2e). The modified residues were distributed (i) along the nucleotide binding region (with N³⁵ located in the protein/protein interface of the interaction of Hsc70 with co‐chaperones of the Bag family and N¹⁵¹ in close proximity to the nucleotide binding pocket), (ii) in the substrate binding region, and (iii) in the lid domain (with N⁵⁸⁴ adjacent to K⁵⁸³, one of the lysine residues known to mediate interaction of Hsc70 with phosphatidylserine on the LE/MVB membrane during eMI (Morozova et al., 2016)) (Figure S2d). We propose that glycation of these residues may affect the ability of Hsc70 to hydrolyze ATP, required for releasing bound substrates, its interaction with the co‐chaperone Bag6, recently shown to co‐operate with Hsc70 in the substrate internalization step of eMI (Krause et al., 2022), or interfere with the docking of Hsc70 at the LE/MVB (Morozova et al., 2016).

Since glycation can have a toxic effect on protein function (Simm, 2013), we analyzed changes in the behavior of Hsc70 in the LE/MVB compartment. To investigate the topology of glycated Hsc70, we incubated isolated LE/MVBs at room temperature with increasing concentrations of trypsin, which only degrades surface proteins (mTOR shown as control in Figure S2e). A larger fraction of the glycated form of Hsc70 was still present after trypsinization, suggesting that it may be more readily internalized into LE/MVBs than unmodified Hsc70 (Figure S2e). Although persistence of glycated Hsc70 upon trypsinization could reflect intrinsic resistance to degradation by the protease, we discarded this possibility because we found that both unmodified and glycated Hsc70 displayed comparable kinetics of degradation in LE/MVBs. Incubation of LE/MVBs at physiological temperature (37°C) revealed a sharp reduction in levels of both the glycated and unmodified form of Hsc70 in old LE/MVB that could be prevented by inhibition of proteolysis, in support of the proposed higher internalization and degradation of both forms of Hsc70 with age (Figure 2f,g). In fact, Hsc70 behavior in old LE/MVBs resembled that of the co‐chaperone Bag6 (Figure 2f,g), previously shown to be internalized, with a fraction of it degraded along with internalized substrates as part of the eMI process (Krause et al., 2022). Stability of another eMI component, the ESCRT protein Vps4, was also reduced in old LE/MVBs, albeit to a lower extent (Figure 2f,g). Whether Vps4 instability is related to the change in Hsc70 with age or is an independent event will require further investigation.

To further investigate possible aberrant protein–protein interactions and/or overall changes in the organization of Hsc70 in LE/MVBs with age, we performed blue‐native electrophoresis of isolated organelles. We detected a high molecular weight protein complex (~970 kDa) immunoreactive for Hsc70 only present in old LE/MVBs (Figure 2h), and two additional Hsc70 complexes (~930 and 600 kDa), also detected in young LE/MVBs but in significantly lower abundance than in old LE/MVBs (Figure 2h,i). The increase in abundance of Hsc70 in these high molecular weight complexes in old LE/MVB was almost 3 times the increase in overall levels of Hsc70 in these organelles (Figure 2a), supporting that at any given time, a larger fraction of Hsc70 in old LE/MVBs is organized in protein complexes in these organelles. We favor a model where instead of the typical transient interactions of Hsc70 with substrates and other eMI components in LE/MVBs, glycated forms of Hsc70 establish more stable interactions and consequently limit the number of sites and/or components available for eMI activity and slows down this process (Figure 2j).

We next used stimulated emission‐depletion (τ‐STED) microscopy, a variety of super‐resolution microscopy, in isolated LE/MVBs to investigate changes with age in the organization and dynamics of Hsc70 and Bag6. After incubation or not of isolated LE/MVBs with Tau protein to stimulate eMI activity, we immunostained for LAMP‐1 to label the LE/MVB membrane, Tau, and the two chaperones (Figure 3a,b). Quantitative tracing of the proteins between the external and internal perimeters of individual LE/MVBs revealed a relatively uniform distribution of Hsc70 along the membrane and areas of discrete Bag6 staining previously described as points of active eMI substrate internalization (Krause et al., 2022) (Figure 3c). Analysis of areas with high signal intensity, or “hot spots” (pixels with intensity >1.25 times the average pixel intensity) showed differences in the overall distribution of Hsc70 and of Bag6 in hot spots in the oldest group, mostly upon addition of the substrate protein (Figure 3d). Although the average distribution of both chaperones did not change significantly upon addition of Tau, we found differences with age in the fraction of LE/MVBs with chaperones preferentially inside or outside the “hot spot” regions. Young mice presented a relatively balanced fraction of LE/MVBs displaying the chaperones outside and inside hot spots, likely representative of substrate binding and internalization sites, respectively (Figure 3e). In contrast, in most LE/MVBs from old mice, both proteins remained in areas of high signal intensity (Figure 3e), compatible with the higher Hsc70 clustering observed using biochemical procedures (Figure 2h). Analysis of the distribution of the chaperones relative to the substrate also revealed lower coincidence of substrate and chaperone in the Hsc70 “hot spots” (Figure 3f) in agreement with our hypothesis that part of the clustered modified Hsc70 was no longer capable of engaging in substrate binding/internalization.

We used the same tracing strategy to identify the fraction of each chaperone found in the lumen of LE/MVBs and the impact of activating eMI (by incubation with Tau) on their internalization. We noticed significantly higher levels of luminal Hsc70 in old LE/MVBs upon addition of Tau (11.9% vs. 21.2%) and a similar pattern for Bag6 (19.8% vs. 29.5%) (Figure 3g). Although a fraction of Hsc70 was also internalized in a substrate‐dependent manner in young LE/MVBs, Hsc70 internalized in the old LE/MVBs was 3‐fold more (78% in 22 m old vs. 26% in 4 m old), thus mirroring our biochemical findings (Figure 3f,g). Using simple linear correlation between Tau internalization and Hsc70 and Bag6 internalization (Figure 3h), we found that the strong correlation between substrate and chaperone internalization noted in young LE/MVBs (slope = 1.052, R² = 0.41, p < 0.0001) was less evident in old LE/MVBs, where higher amounts of chaperones were internalized per molecule of substrate (slope = 0.7303, R² = 0.14, p = 0.037). It is possible that chaperone internalization with age occurs in part in a substrate‐independent manner and/or that the observed clustering of Hsc70 in the old LE/MVBs promotes internalization of multi‐chaperone complexes per substrate in these conditions. In fact, we also noticed some level of dissociation with age in the LE/MVB dynamics of Bag6 relative to Hsc70. We recently found that the ratio of Bag6 internalized for every Hsc70 molecule changes with eMI activity (Krause et al., 2022), with a higher amount of Bag6 to Hsc70 internalized when eMI is activated, as shown here for young LE/MVBs (Figure 3i). However, similar analysis in the old LE/MVBs revealed a loss of the coordinated substrate‐dependent increase in Bag6 internalization with Hsc70 (Figure 3i), in support of altered Bag6/Hsc70 dynamics.

Overall, our findings suggest that reduced eMI with age may be a consequence of altered Hsc70 dynamics at the LE/MVB membrane that lead to its higher internalization and subsequent augmented degradation in this compartment. We attribute the disrupted dynamics to age‐related post‐translational modifications on Hsc70, such as the glycation identified here, that affect the normal interaction of this chaperone with substrates and other eMI components.

Since our analysis of endogenous eMI substrates indicated that the impact of aging on eMI may be, to some extent, substrate‐dependent (Figure 1i,j), we performed comparative quantitative proteomics of isolated LE/MVBs from young and old mice to determine changes in the profile of eMI substrates with age. We injected half of the mice with leupeptin, as in Figure 1g, to inhibit proteolysis in the LE/MVB lumen and thus cause the accumulation of endogenous substrates (proteins with an increase of ≥20% upon leupeptin injection) (Schneider et al., 2014). When proteolysis was not inhibited, more proteins displayed higher levels in LE/MVBs from old mice when compared to young mice (Figure S3a; 29% of the LE/MVB proteome in 22 m old vs. 15% in 4 m old). However, upon leupeptin administration, we detected a clear shift toward accumulation of more proteins in LE/MVBs from young mice and a marked reduction in the number of proteins undergoing degradation in the old (Figure S3b; 39% of the LE/MVB proteome in 4 m old vs. 11% in 22 m old). The more pronounced effect of the proteolysis blockage in young LE/MVBs is in agreement with the observed reduced proteolytic activity of old LE/MVBs (Figure 1k). In fact, under our experimental conditions, we found that 50% of proteins detected in young LE/MVBs were undergoing degradation compared with only 17% of proteins in old LE/MVBs (Figure S3b).

To differentiate substrate proteins reaching LE/MVBs through eMI (from the cytosol), from those degraded in LE/MVBs through heterophagy (pinocytosis, phagocytosis, endocytosis), we used as before (Krause et al., 2022) Gene Ontology (GO) analysis to eliminate proteins associated with the terms “extracellular space” and “plasma membranes”, which included 34% and 25% of the degraded proteins in young and old LE/MVBs, respectively. Analysis of the remaining proteins revealed 813 proteins undergoing degradation in LE/MVBs in young mice and only 334 in old mice (Figure S3c–f). Interestingly, in addition to the 676 proteins no longer degraded in the old LE/MVBs, there was a subset of proteins (197) not previously detected to undergo degradation in this compartment in young mice that were degraded in old mice (Figure S3f). This group included proteins normally present in LE/MVBs as constitutive components that become unstable in this compartment with age (184 proteins), as well as 13 cellular proteins only routed to LE/MVBs for degradation in the old group (Figure S3g). We also identified proteins with defective eMI targeting to LE/MVBs with age as those degraded in young LE/MVBs and no longer detected in the LE/MVBs from old mice injected or not with leupeptin (Figure S3g). Only 7 proteins fulfilled these criteria, further confirming that LE/MVB targeting of eMI substrates is preserved with age and that the primary defect in this autophagic pathway is in internalization/degradation of cargo.

Sequence analysis of proteins degraded in LE/MVBs confirmed similar enrichment in both age groups (~80%) of proteins bearing KFERQ‐like targeting motifs (indicative of selective eMI) (Kirchner et al., 2019) (Figure S3h). These proteins also showed comparable distribution of motifs constitutively present in the protein sequence (canonical) and those that become motifs upon post‐translational modifications, namely phosphorylation or acetylation (Figure S3h). These findings further support that most of the identified cargo reached LE/MVBs in all cases through KFERQ‐selective eMI. To determine the contribution and possible changes with age on the degradation of supersaturated proteins (proteins at risk of aggregation when their cellular concentrations reach their solubility limit), we analyzed the supersaturation scores (Ciryam et al., 2013) of proteins differentially degraded in LE/MVBs with age. We found similar scores for proteins degraded only in young or only in old mice when in their folded state (Figure S3i, left), but we noticed a significantly reduced ability of LE/MVBs from old mice to handle proteins with higher supersaturation scores in the unfolded state (Figure S3i, right). These findings are compatible with the failure of eMI with age contributing to the accumulation of prone‐to‐aggregate proteins often found in old organisms.

To gain information on the possible consequences of the quantitative and qualitative changes with age in the profile of proteins degraded by eMI, we investigated the cellular pathways in which those proteins participate. STRING analysis (Szklarczyk et al., 2019) (Figure S4a,b) revealed that degradation by eMI of proteins involved in other proteolytic systems (proteasome and mitophagy) was lost with age, whereas two major functional groups, ribosome and metabolism, were still enriched as eMI substrates in both ages. However, the fact that the specific proteins in those functional groups were different suggests that eMI may regulate similar pathways in aging, although via the degradation of different substrates, and consequently with different outcomes. To further refine the metabolic processes regulated by eMI in both ages, we used Reactome analysis (Jassal et al., 2020) (Figure S4c–f) and found that eMI predominantly degrades proteins that regulate metabolism of carbohydrates in young mice and those involved in metabolism of lipids, specifically fatty acids, in old mice (Figure S4e,f).

As the LE/MVB is also the site of exosome biogenesis (Stoorvogel et al., 2002), one of the potential consequences of reduced degradation of cargo internalized through eMI in aging could be increased exosome secretion as a way to remove toxic protein products from the cell. In fact, previous studies from our group have shown that this is the case for pathogenic forms of Tau proteins, which have inhibitory effects on different autophagic pathways. These proteins reach the extracellular space in large part through their eMI‐dependent delivery to LE/MVBs and subsequent fusion of these compartments with the plasma membrane (Caballero et al., 2021). To study the relationship between the impairment of intracellular proteolysis and changes in protein secretion with age, we used primary ear fibroblasts from young and old mice. To avoid the confounding effect of elevated secretion in cellular senescence (Coppé et al., 2008), we only used early passage cultures, which we previously confirmed contained less than 1% of senescent cells (positive for β‐galactosidase) (Bejarano et al., 2018). We used a pulse of metabolic labeling with ³H‐leucine and then measured secretion of radiolabeled proteins in the extracellular media of cultured fibroblasts (Figure 4a). We found a significant 73% increase in protein secretion by fibroblasts from old mice compared with young mice (Figure 4b). Interestingly, this increase in secretion can be reproduced in young mouse fibroblasts upon chemical blockage of their endolysosomal degradation with a combination of ammonium chloride and leupeptin (N/L) (Figure 4c, left). The lack of further increase over the already augmented protein secretion in old mouse fibroblasts upon treatment with endolysosomal proteolysis inhibitors (Figure 4c, right) further reinforces that faulty degradation in this compartment may be behind increased protein secretion.

Higher protein secretion in the old fibroblasts was not through conventional secretion because it remained unchanged upon treatment with Brefeldin A (conventional secretion inhibitor) (Ripley et al., 1993) (Figure 4d). In contrast, and in support of the contribution of unconventional secretion mechanisms, we found a markedly higher amount of protein (79% increase) present in extracellular vesicles (ECVs) isolated from the culture media of 22 m old mouse fibroblasts when compared with those from 4 m old mice (Figure 4e). This increase did not result from an increase in total intracellular protein content, which remained unchanged with age (Figure 4e).

nFCM NanoAnalyzer tracking analysis of vesicle size distribution revealed a 50% increase in the number of vesicles obtained in the old ECV fraction compared with the young ECV fraction (Figure 4f), and confirmed a predominant content of vesicles in the 30‐100 nm diameter range, compatible with exosomes (Figure 4g). Despite a higher number of vesicles, immunoblotting of the same amount of total ECV protein showed lower abundance of exosome markers CD63 and Tsg101 as well as of Hsc70 in ECVs from old fibroblasts (Figure 4h,i). We postulated that this reduction may reflect the accumulation of undegraded cargo in the ECVs of the old fibroblasts, which would lower the contribution of integral exosome markers to the total amount of loaded proteins. In fact, normalization of the protein loaded to the amount of the ECV marker Tsg101 confirmed a marked increase in the ratio of cargo to this exosome constituent protein (Figure 4j).

We next used the proteomic analysis of LE/MVBs from young and old mice livers (Figures S3,S4) to identify age‐related changes in the constitutive components of this compartment that could support a higher secretory propensity with age. We first compared the proteomic profile of LE/MVBs intrinsic proteins (those proteins detected in young LE/MVBs whose levels did not change upon inhibition of proteolysis). In the case of old mice LE/MVBs, we eliminated from this list those proteins identified as bona fide substrates in young LE/MVBs, as those represent cargo with compromised degradation with age. This comparison identified changes in 109 of the LE/MVBs resident proteins, with 33 proteins displaying reduced abundance and 76 present at higher levels in old mouse LE/MVBs (Figure 4k). Analysis of LE/MVB resident cathepsins and other hydrolases and of LE/MVB membrane proteins, including subunits of the vacuolar ATPase (V‐ATPase), showed no reduction in their levels with age, and in fact, most of them were more abundant in LE/MVBs from old animals (Figure S3j) in agreement with our immunoblot and enzymatic analysis (Figure S1a–c) and with the higher acidification observed in old LE/MVBs (Figure 1l). When considering all LE/MVB resident proteins, STRING analysis demonstrated enrichment in old LE/MVBs of proteins in functional families related with vesicular trafficking, membrane budding and association to the plasma membrane (Figure 4l), all events that could contribute to LE/MVB‐mediated protein secretion. Examples of proteins related with exosome‐mediated protein secretion that display higher levels in old mouse LE/MVBs are shown in Figure 4m.

To identify possible molecular mediators of the proposed coordinated functioning of eMI and exocytosis, we performed a CRISPR interference (CRISPRi) screen in cells expressing a nuclease‐dead version of the Cas9 enzyme (dCas9) tagged with the KRAB transcriptional repressor and transduced for stable expression of the KFERQ‐split Venus eMI reporter (Figure S5a). This reporter allows visualization of the sequestration of this artificial eMI substrate in the inward budding vesicles at the LE/MVB limiting membrane (Caballero et al., 2018; Krause & Cuervo, 2021). The reporter only fluoresces when the two halves of the Venus protein come in close proximity in the confined space of the intraluminal vesicles of LE/MVBs, making measurement of the number of fluorescent puncta per cell a reliable measurement of eMI. We used a library of short guide RNAs (sgRNAs) targeting proteostasis‐related genes (including genes involved in protein folding, degradation and secretion and in endolysosomal and autophagy pathways [Horlbeck et al., 2016]) and used fluorescent activated cell sorting to separate cells with high (top 33%) and low (bottom 33%) Split Venus signal to determine the frequency of individual sgRNAs in each population by next‐generation sequencing (Figure S5b).

Among the top hits of the screen, we noticed that transcriptional repression of 3 components of the exocyst complex, previously linked to vesicle secretion (Guo et al., 1999; TerBush et al., 1996), led to significant changes in eMI activity (Figure S5c,d). The exocyst is an eight‐member protein complex organized into two different subcomplexes (SCI and SCII) (Figure 5a) (Ahmed et al., 2018), whose assembly and cellular localization are often regulated by the GTPases RalA and RalB (Moskalenko et al., 2003). The exocyst mediates tethering and localization of secretory vesicles to the plasma membrane, but some exocyst components have been reported to be associated with LE/MVBs (Monteiro et al., 2013). We confirmed that members of the two exocyst subcomplexes, as well as RalA and RalB, associate with LE/MVBs (Figure 5b,c).

Using NIH3T3 mouse fibroblasts stably expressing the KFERQ‐split Venus reporter, we analyzed the effect on different eMI steps of individual knock‐down (KD) of three members of exocyst SCI (Exoc2, Exoc3, and Exoc4), three members of exocyst SCII (Exoc5, Exoc6, and Exoc7) or of RalA (Figure S6a,b shows KD efficiency for each protein). We quantified the amount of reporter sequestered in the LE/MVB (binding/internalization) as the number of fluorescent puncta in cells incubated in the absence of protease inhibitors, and the amount of reporter undergoing eMI degradation as the increase in the number of fluorescent puncta per cell upon inhibition of endolysosomal proteolysis with N/L treatment (Caballero et al., 2018; Krause & Cuervo, 2021). We observed only minimal changes in eMI binding and trends to increased degradation upon knockdown of several exocyst subunits in cells maintained in the presence of serum (Figure 5d,e). However, upon serum deprivation, a condition shown to inhibit eMI (Krause et al., 2022), we found significant increases in both eMI substrate binding/internalization and degradation upon knockdown of components of exocyst SCI (Exoc3, Exoc4) and SCII (Exoc5, Exoc6) (Figure 5d,f). Pharmacological inhibition of exocyst activity with Endosidin 2 (ES2) (Zhang et al., 2016) in control cells stably expressing the KFERQ‐Split Venus reporter demonstrated a similar dose‐dependent stimulatory effect on eMI, mainly at the degradation step (Figure S6c). Interestingly, KD of the regulatory component RalA, although less efficient than for other exocyst components, displayed one of the most pronounced stimulatory effects (approx. 10‐fold increase over control cells) when eMI was repressed by serum deprivation, but it lacked any effect over basal (Serum+) eMI (Figure 5d–f), suggesting that RalA may be a key effector of the inhibition of eMI in the absence of nutrients. Overall, these findings are consistent with an inhibitory role for the exocyst complex and the RalA effector in eMI activity.

We next set to elucidate if part of the inhibitory role of the exocyst complex on eMI activity occurred directly at the level of the individual LE/MVB. Trypsinization of isolated LE/MVBs to determine the topology of Exoc4 and Exoc7, as representative examples of components of exocyst SCI and SCII, respectively, showed that the majority of Exoc4 and Exoc7 was present at the surface of LE/MVBs (susceptible to trypsin proteolysis) (Figure 6a). We also detected a small fraction of each protein only degraded upon disruption of the membrane with detergent (Figure 6a), in support of their presence in the lumen of LE/MVBs. This luminal fraction was protected from degradation by LE/MVBs proteases, as it remained unchanged upon incubation of these organelles at 37°C (Figure 6a) and may represent the previously reported presence of exocyst components inside exosomes (Chacon‐Heszele et al., 2014). Activation of eMI by incubation of LE/MVBs with Tau protein did not affect the surface/lumen distribution of exocyst components (Figure 6a), suggesting that internalization of exocyst proteins may be independent of eMI and motivating us to focus on the surface associated exocyst proteins.

We used the in vitro eMI reconstitution assay to investigate the effect on eMI of pre‐incubating LE/MVBs with increasing concentrations of antibodies against Exoc2 (SCI) or Exoc5 (SCII) (Figure 6b; immunoblot confirmed efficient binding of both antibodies to the exocyst proteins to the LE/MVB surface). Antibody blockage of either Exoc2 and Exoc5 did not change Tau binding to LE/MVB but led to a significant dose‐dependent increase in Tau internalization/degradation (Figure 6b–d). These findings are consistent with the data from the cell KD experiments showing a modest effect on eMI binding and a substantial effect on eMI degradation.

Since exocyst complex inhibition of eMI occurs at the level of individual LE/MVBs, we analyzed possible interactions with eMI components in this compartment. Co‐immunoprecipitation for Bag6, Hsc70, Tsg101, or Alix from isolated LE/MVBs revealed that Exoc4 and Exoc7 co‐precipitated with Hsc70 and Bag6 (Figure 6e; RalA and LAMP‐1 are shown in Figure S6d as positive and negative control of Exoc4 interaction, respectively). Although limited by the availability of antibodies suitable for reverse co‐immunoprecipitation, we were able to at least confirm that pull‐down with anti‐Exoc4 co‐immunoprecipitated Hsc70 (Figure S6e). Interaction of Exoc4 or Exoc7 with Hsc70 and Bag6 was not detected in cytosol (Figure 6e), thus making it unlikely that their association in LE/MVBs was a result of Exoc4 or Exoc7 being eMI substrates themselves. Furthermore, none of the exocyst components tested underwent degradation in LE/MVBs (Figure S6f shows levels of Exoc2, 4 and 7 in LE/MVBs from mice injected or not with leupeptin).

Since we did not find major changes in the abundance of exocyst components in LE/MVBs with changes in eMI activity (Figure S6g,h shows comparable levels of Exoc2, Exoc4, Exoc7 and RalA in fed and starved mice, where eMI activity is reduced), we next investigated possible differences in their distribution at the LE/MVB membrane in these conditions. τ‐STED microscopy of isolated LE/MVBs immunostained for LAMP‐1, Hsc70, and Exoc2 (Figure 6f) identified regions of coincidence of Exoc2 and Hsc70 signal along the LE/MVB membrane, with discrete foci of Exoc2 compared with the uniform distribution of Hsc70 (Figure 6g). Given the inhibitory effect of the exocyst complex on eMI and the interaction of Exoc4 with Hsc70 and Bag6, we propose a model where the exocyst complex exerts its inhibitory effect on eMI through sequestration of part of the LE/MVB‐associated Hsc70, thus limiting its contribution to eMI (Figure 6f, lower right scheme).

Given the inhibitory effect of the exocyst complex on eMI, we investigated if changes in this complex with age may underlie some of the reduction in eMI with aging. While overall levels of the tested exocyst components (Exoc2, Exoc3, Exoc4, Exoc5, and Exoc7) did not change with age, total cellular levels and LE/MVB levels of both regulatory components RalA and RalB increased over twofold with age (Figure 7a and S7a). Since RalA and RalB have been shown to regulate localization and assembly of the exocyst complex (Zago et al., 2019), we performed blue‐native electrophoresis of young and old LE/MVBs to investigate the oligomeric status of their exocyst complexes. Immunoblot for Exoc4 (SCI) and Exoc7 (the only component of SCII for which we found antibodies able to recognize the native protein) revealed increased abundance of high molecular weight complexes (approximately 900 kDa for Exoc4 and 1020 KDa for Exoc7) in 22 m old LE/MVBs (Figure 7b,c black arrow). The lower molecular weight complex for both proteins (green arrow) may indicate the four‐member exocyst subcomplexes, whereas the high molecular weight band may represent homo or hetero octameric exocyst complexes (~734 kDa) bound to additional proteins (Ahmed et al., 2018). In fact, the Exoc4 high molecular weight complex detected in old LE/MVBs was also positive for Hsc70 (Figure 7d) and coincided in size with the Hsc70‐positive protein complex that we described to be present mostly in old LE/MVBs (Figure 2h). A fraction of Vps4 (Figure 7d), RalA and other components of SCI such as Exoc3 (Figure 7e,f) were also detected in this same high molecular weight region in higher abundance in old LE/MVBs, in further support of disrupted dynamics of both the exocyst complex and eMI machinery with age. Although resolution of the precise size of the different complexes for each of these proteins will require more accurate procedures, our blue‐native electrophoresis studies support coincidence of subunits of SCI, RalA, and Hsc70 in similar size complexes (in the 890–950 kDa range), whereas Exoc7 preferentially distributed in a higher molecular weight complex (1020 kDa) along with a small fraction of Exoc4 (Figure S7b shows the high molecular weight region in more detail).

To confirm direct interaction of the exocyst/RalA components with Hsc70 in LE/MVBs and to determine if the Hsc70 observed in these complexes was the glycosylated form of the chaperone, we performed co‐immunoprecipitation experiments in LE/MVBs. We found that glycosylated Hsc70 co‐precipitated with both Exoc4 (Figure 7g, left) and RalA (Figure 7h) (note that the unmodified hsc70 is barely visible to avoid saturation of the glycosylated form). The presence of glycosylated Hsc70 in the co‐immunoprecipitated fractions was not a contamination because it was not observed, for example, in similar pull‐down experiments for Exoc6 (the only subunit of SCII with antibodies suitable for immunoprecipitation), although Exoc6 was still interacting with a fraction of LE/MVB Exoc4 (Figure 7g right and 7i). These findings are in agreement with the blue‐native electrophoresis data, that placed hsc70 mostly in RalA/SCI complexes (Figure 7d,f and S7b).

Since a fraction of Hsc70 in LE/MVBs interacts with exocyst SCI (Figure 7g) and our knock‐down and τ‐STED experiments suggest that this interaction may be inhibitory on eMI, we postulated that the age‐dependent glycation of Hsc70 may change Hsc70/exocyst SCI dynamics at the membrane of old LE/MVBs and contribute to reduced eMI. Analysis of the distribution of Hsc70 and Exoc2 in isolated 22 m old LE/MVBs using τ‐STED microscopy (Figure 7j,k) and comparison with their distribution in 4 m old LE/MVBs (Figure 3a–c), revealed that Exoc2 is more diffusely distributed in the membranes of old LE/MVBs and displays higher overlap with Hsc70. We propose that sequestration of a higher fraction of LE/MVB‐Hsc70 by the exocyst SCI may decrease the availability of free Hsc70 for eMI with age (Figure 7j, lower right scheme).

To further characterize the changes in association of exocyst SCI and SCII within individual LE/MVBs with age, we performed immunofluorescence for Exoc2 and Exoc7, as representative members of each subcomplex, in spotted LE/MVBs previously incubated with Tau protein (to identify eMI‐active LE/MVBs as those positive for LAMP‐1 and the substrate Tau). We found that the fraction of eMI‐competent LE/MVBs that contained Exoc2 significantly increased with age (18.3% in 4 m old vs. 33.5% in 22 m old) while those containing Exoc7 remained unchanged (9% in 4 m old vs. 11.5% in 22 m old) (Figure S7c,d). These findings suggest that the association of exocyst SCI to eMI‐competent LE/MVBs changes with age and that, because the overall abundance of Exoc2 in LE/MVBs does not change with age (Figure 7a), the increased colocalization of Exoc2 with eMI‐competent LE/MVBs may result from its redistribution from LE/MVBs not engaged in eMI. Immunostaining for RalA in spotted LE/MVBs processed in the same conditions revealed a very marked age‐related increase in the association of RalA with LAMP‐1⁺/Exoc7⁺ vesicles (from 17.6% in 4 m old to 53.8% in 22 m old) (Figure S7e). These findings are in agreement with the increase in RalA levels in LE/MVBs with age (Figure 7a) and higher abundance of octameric exocyst complexes (Figure 7b‐f), which could mediate the observed bias with age of LE/MVBs from degradative to secretory compartments (Figure 4).

The pronounced physiological inhibitory effect of RalA on eMI, whereby a 30% reduction in RalA levels led to >10‐fold increase in eMI degradation (Figure 5d–f), suggests that even small changes in RalA levels can have a marked impact on eMI activity and made us consider that the increase in RalA cellular levels with age (Figure 7a) could be a key contributor to the decline of eMI in aging. To investigate the mechanism(s) behind increased RalA levels in aging, we first analyzed possible transcriptional upregulation, but qPCR analysis revealed instead a significant reduction with age in RalA mRNA (Figure 7l). We instead considered changes in RalA degradation with age and noticed the presence of a KFERQ‐like motif in its sequence (¹⁹⁷KRIRE²⁰¹ becomes a motif upon acetylation of the lysine residue). Since our proteomic analysis (Figure 2 and S7f) did not support degradation of RalA in LE/MVBs and given that KFERQ pentapeptides are shared as targeting motifs for both eMI and CMA (Krause & Cuervo, 2021), we investigated if RalA could be degraded by CMA. We found that a fraction of RalA could be detected in CMA active lysosomes and that in vivo injection of leupeptin resulted in an accumulation of RalA in these lysosomes (Figure 7m), a behavior characteristic of CMA substrates (Hexokinase is shown as an example of a validated CMA substrate). Using LE/MVBs isolated from livers of CMA‐deficient mice (knock‐out for the limiting CMA component LAMP‐2A), we confirmed that blockage of CMA‐dependent degradation of RalA led to significantly higher association of RalA with LE/MVBs (Figure S7g) and led to an accumulation of RalA in total liver homogenate from LAMP‐2A KO mice (Figure S7h). Conversely, pharmacological activation of CMA in old mice, where CMA activity is markedly reduced (Cuervo & Dice, 2000), normalized RalA to the levels observed in young mice. Daily oral administration of CA77.1, a novel, specific CMA activator suitable for in vivo use (Bourdenx et al., 2021) in old mice significantly reduced the age‐related accumulation of RalA without affecting levels of Exoc2 (SCI) or Exoc7 (SCII) (Figure 7n).

Overall, our findings are consistent with an inhibitory role of the exocyst complex in eMI activity and with RalA‐dependent changes with age in exocyst complex dynamics in LE/MVBs, likely as a result of its interaction with glycosylated Hsc70, that result in an inhibitory effect on eMI activity (Figure). Defective degradation of RalA by CMA provides a basis for the age‐related accumulation of RalA and places CMA as a key regulator of eMI activity. The age‐related increase in the organization of exocyst proteins into complexes may reflect both a mechanism for the reduced eMI activity observed in aging and a link between eMI and protein secretion when substrates can no longer be effectively degraded by the pathway (Figure). S8 S8

This study used adult (3–6 months) male Wistar rats (Charles River Laboratories) and male young (4–6 months) and old (22–24 months old) mice (NIA colony), and mice with systemic knockout for LAMP‐2A (L2A^−/−) (Schneider et al., 2014). All animal experiments were under an animal study protocol approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine. In vitro studies were done using NIH3T3 mouse fibroblasts from the American Type Culture Collection (ATCC) or mouse primary ear fibroblasts from 4 m and 22 m old mice, isolated as described previously (Varela et al., 2005). Knock‐down of the proteins of interest was performed using lentivirus containing shRNAs listed in Table S1. Details on animal maintenance and treatments and on cell culture conditions and in vitro treatments are provided under Extended Experimental Procedures (Appendix S1).

Sources of chemicals and dilutions and sources of antibodies used in this study are detailed under Extended Experimental Procedures (Appendix). S1

Rodent liver lysosomes and LE/MVBs were isolated through differential centrifugation and separation into discontinuous density gradients. Cytosol was prepared by centrifugation at 100,000×g for 1 h of the post‐nuclear supernatant. Extracellular vesicles (ECVs) were isolated from the culture media by sequential centrifugation and concentration through molecular exclusion columns. LysoSensor Yellow/Blue DND‐160 was used for pH determination, PNGase F and Endo H for deglycosylation, trypsinization to determine protein topology and incubation at physiological temperature and immunoblot to determine protein stability as described in detail under Extended Experimental Procedures (Appendix S1).

In vitro analysis of eMI was performed using isolated LE/MVBs and purified substrate proteins as described before (Krause & Cuervo, 2021). Binding was calculated as the amount of protein associated with LE/MVBs untreated with protease inhibitors and internalization/degradation as the difference between proteins present in organelles treated with protease inhibitors after subtracting for the amount bound. Proteolysis in isolated LE/MVBs was assayed using a pool of radiolabeled proteins as before (Sahu et al., 2011). Measurement of eMI in cultured cells was done using the KFERQ‐Split Venus reporter (Caballero et al., 2018) described in detail under Extended Experimental Procedures (Appendix S1).

Protein secretion was measure upon ³H‐leucine metabolic labeling of cultured cells by analysis of radioactivity on the acid precipitable fraction collected from the culture media. Co‐immunoprecipitation was performed upon solubilization in a mild detergent/low salt buffer using primary antibodies and Protein A/G Plus agarose beads. Protein electrophoresis and immunoblot, quantitative proteomics, and protein pathway analysis and glycoproteomics were performed using standard procedures described in detail under Extended Experimental Procedures (Appendix S1).

Immunofluorescence was performed in glass spotted organelles, fixed, and incubated with primary and secondary antibodies following standard procedures. STED microscopy of isolated organelles was performed in a Leica TCS SP8 STED 3X outfitted with a τ‐STED module and analysis of membrane/lumen distribution and organization of proteins in hot spots based on protein abundance were performed as described before (Krause et al., 2022) and detailed under Extended Experimental Procedures (Appendix S1).

CRISPR interference (CRISPRi) screen with an sgRNA library targeting 1176 genes related to proteostasis pathways was performed using cells expressing nuclease‐dead form of Cas9 fused to the transcriptional repressor KRAB (dCas9‐KRAB) and the KFERQ‐Split Venus fluorescent eMI reporter as described in detail under Extended Experimental Procedures (Appendix S1). Quantitative RT‐PCR was performed after reverse transcription of RNA into cDNA using the primers shown in Table S2 following isolation of total RNA using the RNeasy Plus kit (Qiagen) according to the manufacturer's instructions.

All data presented are mean ± SEM and individual values. Detailed description of the statistical analysis and software used for acquisition, analysis and presentation of data can be found under Extended Experimental Procedures (Appendix). Raw data and statistical analyses from data presented in main and supplementary figures are provided as a Raw Data Excel file (Appendix) and uncropped images of the unprocessed immunoblots shown in all figures of the manuscript are compiled as figure in Appendix. S1 S1 S1

There are no restrictions on data availability in this manuscript. All the information is included in the manuscript. All Main and Supplementary Figures have associated raw data that are provided as an Excel worksheet organized by figures and it includes statistics along with exact p‐values. The raw data of the proteomic analysis of LE/MVBs have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD035128. Mass spectrometry raw files for the glycosylation analysis have been uploaded on the public repository Chorus (https://chorusproject.org↗) at the project number 1779. This manuscript does not report original code. Further information and requests for reagents may be directed to and will be fulfilled by the co‐corresponding author Ana Maria Cuervo (ana-maria.cuervo@einsteinmed.edu) upon completion of a Material Transfer Agreement.