A RAB7A Phosphoswitch Coordinates Rubicon Homology Protein Regulation of PINK1/Parkin-Dependent Mitophagy

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a cytoprotective mitophagy response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding protein that acts as a negative regulator of autophagy. The structure of the Rubicon-RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon-RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.


Introduction
Macroautophagy (hereafter autophagy) is an ancient and conserved system whereby cells isolate, degrade and recycle cytosol, aggregates, and organelles in response to starvation or stress 1 . Autophagy may take up bulk cytosol non-selectively, or may be selective for particular types of cargo 2,3 . Autophagy is central to neuronal homeostasis, and dysfunction in autophagy is implicated in neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) 4 . Autophagy protects neurons by clearing aggregation-prone proteins (aggrephagy) and damaged mitochondria (mitophagy) 5 . The PD gene products PINK1 and Parkin represent the bestcharacterized example of a disease-linked selective autophagy pathway 6 . The protein kinase PINK1 and the ubiquitin E3 ligase Parkin initiate mitophagy in response to mitochondrial damage 7,8 . The importance of this process in PINK1/Parkin-associated PD has led to an intense focus on understanding the regulation of PINK1 and Parkin 6 , as well as the events of mitophagy initiation that occur immediately downstream of PINK1 and Parkin 5 . PINK1/Parkin mitophagy initiation entails the activation of Parkin, which catalyzes the addition of short ubiquitin chains to mitochondrial outer membrane proteins. These ubiquitin chains are recognized by selective autophagy adaptors, including NDP52 and OPTN [9][10][11] . NPD52, OPTN, and other cargo receptors [12][13][14] then act to recruit the core autophagy machinery, which is broadly conserved among bulk and selective autophagy pathways 15 . The core autophagy machinery includes the Unc-51-like autophagy activating kinase (ULK1) complex, the class III phosphatidylinositol 3-kinase complexes I and II (PI3KC3-C1 and -C2), the PI3P-binding WIPI proteins, the ubiquitin-like ATG8 proteins, the ATG12-ATG5-ATG16L1 complex and associated machinery of ATG8 conjugation, and the ATG2 and ATG9 proteins involved in lipid transfer and autophagosome expansion 1,16 . One of the most critical steps in the pathway is the generation of PI3P by PI3KC3. Membrane recruitment of the WIPI proteins is gated by the presence of PI3P 17 . The conjugation of the ATG8 family proteins and recruitment of the ATG2 lipid transporter for autophagosome expansion depend on the presence of their cognate WIPI proteins 18,19 . This in turn depends entirely on the production of PI3P by PI3KC3.
The centrality of the PI3KC3 complexes to autophagy initiation renders their tight regulation indispensable. PI3KC3-C1 and C2 share the subunits VPS34, VPS15, and BECN1. PI3KC3-C1 uniquely contains the fourth subunit ATG14, while PI3KC3-C2 contains UVRAG. PI3KC3-C1 is uniquely involved in autophagy, while C2 is involved in both endosome biogenesis and autophagy. Rubicon is a negative regulator of PI3KC3-C2 20,21 . Rubicon consists of an N-terminal RUN domain 22 , a central intrinsically disordered region that includes the PI3KC3-C2 binding domain 23 , and a C-terminal Rubicon Homology (RH) domain responsible for binding to RAB7A 24,25 . Consistent with its role as a negative regulator of a core autophagy complex, Rubicon knockdown or knockout correlates with resistance to neurodegenerationassociated phenotypes. Genetic ablation of Rubicon improves the resistance of mice to formation of seeded a-synuclein aggregates that are characteristic of PD, and blunts the locomotive dysfunction that typifies aging in Drosophila 26 . Rubicon knockout also improves non-neuronal phenotypes, including resistance to interstitial kidney fibrosis 26 and nonalcoholic fatty liver disease 27 in mice. Conversely, upregulation of Rubicon is associated with neurodegenerative disease. Its expression is upregulated in the spinal cords of sporadic ALS patients 28 .
Rubicon is a member of protein family defined by the presence of a C-terminal RH domain that includes two other autophagy regulators, PLEKHM1 24 and Pacer 29,30 . Despite the presence of an RH domain in both proteins, Pacer appears to act in direct opposition to Rubicon, activating PI3KC3-C2 rather than inhibiting it ( Figure 1A) 29,30 . Elevated Rubicon expression in the spinal cords of sporadic ALS patients is paralleled by reduced Pacer expression 28 . Knockdown of Pacer expression via shRNA impairs bulk autophagic flux and the clearance of pathogenic protein aggregates 28 . Thus, Rubicon and Pacer are structurally related proteins that nevertheless appear to play opposing roles in both the cell biology of autophagy and the pathophysiology of neurodegeneration.
Here, we show that binding of RAB7A to the RH family proteins Rubicon and Pacer constitutes a bidirectional switch. Under basal conditions, Rubicon binds unphosphorylated RAB7A and antagonizes autophagy. When RAB7A is phosphorylated following mitochondrial depolarization, Rubicon is displaced, Pacer is recruited, and mitophagy is thereby accelerated. This switch is governed by structural differences in the RH domains of Rubicon and Pacer that have evolved to selectively bind dephospho-RAB7A and pSer72-RAB7A, respectively.

Rubicon inhibits Parkin-dependent mitophagy
The negative regulatory role of Rubicon in starvation-induced autophagy and xenophagy has been well-characterized 20,24 , but it is unknown if Rubicon regulates PINK1/Parkin-dependent mitophagy. Rubicon KO HeLa cells were generated ( Figure S1A, B) and stably transfected with either LC3B-HaloTag to examine bulk autophagy or HA-Parkin and Su9-GFP-HaloTag to examine Parkin-dependent mitophagy. Activation of autophagy induces the engulfment of these reporters within autophagosomes, trafficked to lysosomes, and degraded. In order to initiate the assay, cells were pulsed with membrane permeable fluorescent HaloLigand, which binds HaloTag and renders it resistant to lysosomal processing 38 . Bulk autophagy was induced by incubation in Earle's Balanced Salt Solution (EBSS). Mitophagy was induced by treatment with the electron transport chain inhibitors Oligomycin and Antimycin A (OA), Autophagy flux was measured as the fraction of the pulsed reporter digested within a given interval.
Rubicon knockout strongly increased the extent of both starvation-mediated (by 55%) and basal autophagy (by 47%) ( Figure 1A, B), consistent with past reports 21,26 . We found that Rubicon knockout also increased mitophagy flux, though to a lesser degree than for starvationinduced autophagy (23% increase in mitophagy flux over 6 h of depolarization)( Figure 1C, D). The significantly smaller impact of Rubicon KO on mitophagy flux suggests that there might be a mechanism that renders mitophagy more resistant to Rubicon inhibition than starvationinduced autophagy.

Phosphorylation of RAB7A Ser72 controls binding to RH domains
The RH domain of Rubicon binds to RAB7A, and RAB7A phosphorylation at Ser72 is required for efficient mitophagy flux. We noticed that in the crystal structure of the Rubicon RH-RAB7A-GTP complex, Ser72, part of the GTP-dependent switch II region of RAB7A, is in direct contact with a sterically confined region of the RH domain 25 . We therefore asked whether the phosphorylation state of RAB7A modulated Rubicon binding. We also considered the possibility that RAB7A phosphorylation might regulate Pacer, since Pacer also contains an RH domain ( Figure 2A). However, our structural modeling suggested that the analogous region of interaction with RAB7A is less sterically confined.
To probe these interactions, we expressed and purified recombinant Rubicon and Pacer RH domains, RAB7A, and one of the key kinases known to phosphorylate RAB7A, TBK1 36 . Incubation of RAB7A with TBK1 in the presence of ATP resulted in stoichiometrically phosphorylated pSer72 RAB7A. Assay of this material using a PhosTag gel, which slows the migration of phosphorylated proteins and resolves distinct phosphorylated and unphosphorylated bands, confirmed that the kinase reaction proceeded essentially to completion ( Figure 2B). To assay for RAB7A binding by purified RH domains, we used a confocal microscopy-based bead binding assay. Amylose beads were pre-loaded with MBP-RH domains, and incubated in a solution of the Alexa Fluor 647-labelled GTP-locked RAB7A Q67L ( Figure 2C). The Rubicon RH domain robustly bound unphosphorylated RAB7A, but did not observably bind phosphorylated RAB7A. The opposite was true for the Pacer RH domain. Pacer-RH robustly bound only to Ser72 pRAB7A, with little to no binding observed to unphosphorylated RAB7A. These findings were confirmed by pulldown assays ( Figure S2A). We followed up on these results, measuring the binding affinity of Rubicon RH for RAB7A via isothermal titration calorimetry (ITC). This binding interaction is characterized by a KD of 1.7 ± 0.3 µM ( Figure 2F). This represents a moderate affinity interaction, consistent with a regime where phosphorylationinduced affinity changes could effectively regulate the interaction between Rubicon and Pacerbinding competent states for RAB7A.
To attempt to explain why phosphorylation of RAB7A has opposing effects on binding to the Rubicon and Pacer RH domains, homology modelling was used to generate predictions for the Pacer-pRAB7A binding interface ( Figure 2E). These models were then compared to the crystal structure of the Rubicon RH-RAB7A complex 25 . The Pacer RH domain structure has a prominent basic patch that is absent in the Rubicon RH domain, and appears poised to bind the pSer72 phosphate. Basic residues Lys534, Arg623, and Arg628 within Pacer are all close enough to the negatively charged pSer72 phosphate to form direct salt bridges. In contrast, the Rubicon crystal structure shows that phosphorylation of Ser72 creates a steric clash by introducing the bulk of the phosphate group into a constricted site 25 .

Phosphorylation of RAB7A Ser72 controls subcellular localization of RH proteins
In order to determine whether the in vitro binding preferences were correlated with subcellular ocalization, we transfected Parkin-expressing HeLa cells with mCherry-Rubicon, and performed immunofluorescence using anti-RAB7A and anti-pSer72-RAB7A (Abcam ab302494) ( Figure 3A, B). In these cells, Rubicon clusters near the nucleus and co-localizes with RAB7A. In contrast, pSer72 RAB7A puncta were found throughout the cytoplasm and colocalized only sporadically with Rubicon. We found that Rubicon is more than three-fold more likely to colocalize with RAB7A than with pSer 72 RAB7A, consistent with the in vitro finding that RAB7A phosphorylation negatively regulates Rubicon binding ( Figure 3C).

A RH domain basic triad controls pRAB7A binding
In order to test the structural model for phosphorylation-dependent RAB7A binding, we generated a triple Rubicon RH mutant that contains the Pacer basic triad at the equivalent residues (Rubicon N821K, T911R, K916R), and performed the confocal bead binding assay using these mutants. The Rubicon mutant recruited pRAB7A to beads more effectively than it recruited RAB7A to beads ( Figure 3A). Overall, it appears that this mutation enabled Rubicon to bind pRAB7A at the cost of a moderate decrease in its ability to bind RAB7A ( Figure 3B). This suggests that the Pacer basic triad is responsible for conferring binding to pSer72 RAB7A. Reciprocally, mutants of the Pacer RH domain that swap these residues to their Rubicon equivalents (K534N, R623T, R628K) were generated, purified, and assayed in the fluorescent bead binding assay. The recruitment of pRAB7A was sharply diminished by even one of these mutations, while the double and triple mutants resulted in nearly complete loss of pRAB7A binding ( Figure 4A). These mutations did not, however, confer on Pacer the ability to bind dephosphorylated RAB7A.
To determine the role of the RAB7A phosphoswitch on the localization of Pacer in cells, we compared the colocalization of wild-type and mutant Pacer with pRAB7A. Stable Parkinexpressing HeLa cells were transiently transfected with mCherry-tagged Pacer or K534N/R623T Pacer, fixed, and imaged by confocal microscopy ( Figure 4C, D). Pacer formed sparse punctate structures evenly distributed throughout the cell, in contrast to the perinuclear localization of Rubicon. Pacer co-localized extensively with pRAB7A (40%), while only about 10% of Pacer K534N/R623T did so ( Figure 3E), demonstrating that these residues are critical for proper Pacer localization. These data show that both Rubicon and Pacer binding to, and colocalization with, RAB7A is controlled by the phosphorylation state of RAB7A Ser72, which is in turn subject to induction by the same conditions that induce mitophagy.

Pacer regulates PINK/Parkin-dependent mitophagy
Pacer was reported to upregulate starvation-induced autophagy 29 , but roles in selective autophagy have not been reported on in the literature. To compare the roles of Pacer in bulk autophagy and mitophagy, Pacer knockout HeLa cells were generated via CRISPR-mediated deletion ( Figure S1C), and the autophagy and mitophagy flux reporters LC3B-and Su9-GFP-HaloTag were stably transfected to probe bulk autophagy and mitophagy.
Pacer knockout HeLa cells displayed significantly reduced mitophagy flux following mitochondrial depolarization with OA, (Figure 5A, B). To probe the contribution of pRAB7A binding to Pacer activity, Parkin and mitophagy reporter-expressing cells were stably transfected with vector, wild-type Pacer, and Pacer K534N, R623T . These cells were depolarized for 2 and 4 hours, and mitophagy flux was measured. Rescue with WT Pacer was evident after 2 hours, and mitophagy flux after 4 hours increased by up to 47% relative to KO cells. In contrast, the Pacer K534N, R623T mutant was unable to promote mitophagy flux above the levels seen in Pacer KO cells, suggesting that pRAB7A binding is indispensable to the mitophagy-promoting activity of Pacer ( Figure 5C, D).
To determine if Pacer might have a role in Parkin-independent mitophagy, these same cells were treated with the iron chelator deferiprone (DFP). DFP-mediated mitochondrial iron loss results in increased expression of mitophagy receptors including NIX and BNIP3 through HIF1a signaling [39][40][41] . There was no discernible difference between Pacer KO cells with vector control or wild-type or mutant Pacer at 8, 16, or 24 H ( Figure 5E). This indicates that Pacer has a specialized role in PINK1/Parkin-dependent mitophagy.
The impact of Pacer and pRAB7A-dependent action on starvation autophagy was assayed by creating stable transfectants of wild-type Pacer and Pacer K534N, R623T in Pacer KO HeLa cells expressing the LC3B-Halo bulk autophagy reporter. In contrast to the previous report of a Pacer role in starvation autophagy 29 , these cells showed negligible change in the level of starvationinduced bulk autophagy flux at any time point tested. This indicates that, at least in HeLa cells, Pacer is principally an activator of PINK1/Parkin mitophagy rather than of autophagy generally ( Figure 5F, G).

Pacer upregulates a pre-fusion stage of mitophagy
Pacer is thought to potentially promote autophagy via two mechanisms. First, Pacer is known to bind PI3KC3-C2 29 through a domain also present in Rubicon 23 . Second, Pacer has been proposed to promote recruitment of the HOPS complex to sites of autophagosomelysosome fusion 29 . HOPS complex recruitment to promote autophagosome-lysosome fusion is also dependent on RAB7A 42,43 . While HOPS functions at the last stage in the progression of autophagy, PI3P production is essential throughout the initiation and expansion stages preceding fusion with lysosomes. To determine whether the pRAB7A-binding dependent action of Pacer occurs early or late in mitophagy, we imaged mCherry-tagged wild-type and mutant Pacer expressing cells using confocal microscopy. We quantified autophagosome formation and autophagosome/lysosome colocalization using live-cell imaging. Autophagosomes were monitored with transfected HaloTag-LC3B and lysosomes were tracked via transfection with LAMP1-mNeon green ( Figure 6A).
We found that the number of wild-type Pacer puncta per cell ranged from 2 to 10 puncta under nondepolarizing conditions, but increased as much as 3-fold following mitochondrial depolarization using CCCP. To contrast, the number of mutant Pacer puncta was insensitive to mitochondrial depolarization ( Figure 6B). Pacer localized robustly with LC3B ( Figure 6A). Expression of wild-type Pacer during OA treatment increased the total surface area positive for LC3B in the cell by 58% ( Figure 6C). The total autophagosome-lysosome colocalized area also increased by 70% upon WT Pacer expression ( Figure 6D), roughly proportionate to the increase in the LC3B positive area alone. This implies that the increased mitophagy flux that characterizes Pacer expression is primarily due to an increase in autophagosome formation rather than delivery to and fusion with lysosomes. If Pacer were principally a promoter of fusion in mitophagy, we would have expected the total LCB3-positive area to decrease as a result of Pacer expression due to accelerated fusion and degradation. Additionally, as an index of autophagosome-lysosome fusion efficiency, we quantified the fraction of the autophagosome area that is also positive for lysosomal markers on a per cell basis. Here, we found that there were only minor differences in fusion efficiency across the KO, wild-type, and mutant conditions. Wild-type Pacer trends towards significance in promoting fusion (p =0.07), suggesting that Pacer may have fusion-promoting activity, but this likely plays only a minor role in mitophagy promotion in this system ( Figure 6E). Pacer thus promotes mitophagy at the level of initiation and/or expansion, consistent with PI3K activation. In contrast to wild-type Pacer, transfection with the Pacer K534N, R623T did not significantly increase the LC3B-positive surface area nor the LC3B-LAMP1 colocalized area.

Discussion
Here, we uncovered an unexpected and elegant mechanism whereby a single RAB phosphorylation event leads to dissociation of an inhibitor of mitophagy and recruitment of an activator. The roles of a variety of molecules in mitophagy, including RAB7A, Pacer, and Rubicon, have thus been put into new perspective. RAB7A is not known to be phosphorylated under starvation conditions. In the absence of RAB7A phosphorylation, Rubicon remains a stronger inhibitor of starvation-mediated autophagy. Pacer, however, appears to be a potent and specific PINK1/Parkin-dependent mitophagy accelerator, whilst having a minimal role in starvation-induced autophagy and DFP-induced mitophagy. This is consistent with the strong dependency of Pacer action on RAB7A phosphorylation. Here, we found that PINK1/Parkin mitophagy increases less in response to Rubicon KO than starvation autophagy. The difference can be explained by the ability of RAB7A phosphorylation to abolish binding of Rubicon to RAB7A, which removes the Rubicon blockade upon mitophagy induction.
Phosphorylation of RAB proteins, especially in switch II at residues equivalent to RAB7A Ser72, is a frequent point of regulation in the endocytic system 33,44 . The discovery that the LRRK2 kinase mutated in PD acts by phosphorylating RAB proteins has spurred intense effort to characterize the downstream effects of RAB phosphorylation 33 . RAB7A is not a known substrate of LRRK2, rather it appears to be a substrate of at least two other protein kinases, TBK1 and LRRK1. TBK1 is well-established as a central player in PINK1/Parkin mitophagy [9][10][11] , and a mitophagy role for LRRK1 was more recently reported 35 . The best explored function of RAB phosphorylation in autophagy is the regulation of JIP4-dependent transport of mature autophagosomes in axons by LRRK2 phosphorylation of RAB29 45 . Here, we have discovered that RAB phosphorylation can also regulate the process of autophagosome biogenesis itself.
It was previously reported that pSer72 RAB7A preferentially recruits the FLCN-FNIP1 complex relative to unphosphorylated RAB7A 36 . FLCN:FNIP1/2 is a GTPase activating protein complex (GAP) for the RagC/D GTPases involved in activating mTORC1 to phosphorylate and inactivate the lysosomal and autophagic transcriptional regulator TFEB [46][47][48] . The FNIP1containing FLCN:FNIP isocomplex is centrally involved in sensing and responding to changes in mitochondrial metabolism 49,50 . FLCN KO does not phenocopy the effect of pRAB7A phosphorylation on recruitment of ATG9A to mitochondria 36 , a key step in mitophagy initiation.
Thus, it was postulated that RAB7A phosphorylation must have additional effects on the autophagy machinery. This concept is consistent with our own finding that purified FLCN-FNIP1 binds robustly to dephosphorylated RAB7A in vitro (unpublished data). While the possibility of yet other effects of RAB7A on the mitophagy machinery cannot be excluded, RAB7A Ser72 phosphoregulation of Rubicon and Pacer is, in principle, sufficient to account for the pRAB7A dependence of PINK1/Parkin mitophagy.
Most models for the regulation of selective autophagy focus on the first step, substrate recognition by cargo receptors 2,3 . The assumption is often made that once initiated, the downstream cascade of protein interactions in autophagosome biogenesis is the same in bulk autophagy and the various forms of selective autophagy. Recent evidence shows that the situation is more complicated. In OPTN-dependent mitophagy, for example, the order of recruitment of the core autophagy components ULK1 and PI3KC3-C1 is inverted compared to most other forms of autophagy 37 . Now we have uncovered a PINK1/Parkin mitophagy-specific mode of regulation of PI3KC3-C2 via Rubicon and Pacer, suggesting that selective autophagy subtype-specific regulator mechanisms may permeate the entire process of autophagosome biogenesis. The existence of these mechanisms may provide new opportunities for the modulation of specific types of autophagy for therapeutic benefit.

Structural modeling
In order to generate a homology model for the Pacer RH domain, the Pacer RH sequence (corresponding to residues 412 through 654) in FASTA format was uploaded to the Biozentrum Swiss-Model. This sequence was aligned against the Rubicon sequence, and a model generated using a published crystal structure of the Rubicon RH domain in complex with RAB7A (PDB: 6wcw 25 ).

Plasmid Construction
In order to generate MBP-tagged RH domains for purification and subsequent in vitro binding experiments, the RH domain-encoding sequence of Rubicon and Pacer were amplified via PCR, purified via gel electrophoresis using a 1% agarose gel, and then subcloned into pET His10 MBP Asn10 TEV LIC cloning vector (2CT-10) via ligation independent cloning (LIC). XL10 Ultracompetent E. coli (Agilent technologies) were transformed with this plasmid, and then plated on ampicillin selective agar. Individual colonies were picked via a sterile loop and grown overnight in 5 mL of LB supplemented with ampicillin. Purified plasmid DNA was isolated via a commercial spin miniprep kit (Qiagen), and cloning was confirmed via Sanger sequencing.
To generate point mutants of the Pacer RH domain, 'round-the-horn site-directed mutagenesis was employed. Briefly, non-overlapping primers, adjoining each other and "pointing in the opposite direction" are designed. One of the primers contains the desired mutations, and the primers are phosphorylated using polynucleotide kinase prior (NEB) to PCR such that each PCR product strand can be immediately ligated to itself. Following PCR using Q5 polymerase (NEB), the product is gel-purified on a 1% agarose gel, and ligated via incubation with T4 DNA ligase (NEB) prior to transformation into XL10 E. coli. The N821K, T911R, K916R mutant of the Rubicon RH domain was synthesized by Genscript in a pMAL-c5E vector.
In order to generate mCherry-tagged WT Rubicon and Pacer for use in confocal imaging and autophagy flux experiments, primers were designed to amplify the sequence encoding fulllength Rubicon and Pacer respectively, using existing Rubicon and Pacer plasmids as templates 23 while incorporating a BglII restriction site upstream of the open reading frame and a stop codon followed by a SalI restriction site downstream of the open reading frame. Following PCR amplification, the product, as well as a stock of pmCherryC1 mammalian expression vector (Clontech) was digested using BglII and SalI (NEB) according to manufacturer instructions. The digested PCR product and vector were purified via electrophoresis on a 1 and 0.5% agarose gel respectively. Subsequently, the Rubicon/Pacer full-length sequence was ligated into the pmCherry-C1 vector via an overnight incubation with T4 DNA ligase (NEB) at 16 °C.
RH domain mutants of full-length Pacer were generated via Gibson Assembly. Briefly, a fragment consisting of Pacer residues 1-412 was amplified via PCR, with a 20 base pair overlap with the pmCherryC1 sequence upstream of the amplified sequence via primer, and a 20 bp overlap with the RH domain included downstream of the fragment. Simultaneously, the mutant RH domains in the 2CT-10 cloning vector were amplified via PCR, with an additional 20 base pair overlap with pmCherryC1 included downstream of the Pacer RH sequence, and a 20 bp overlap with the Pacer 1-412 transcript included upstream. pmCherryC1 was linearized via incubation with BglII and SalI, and then the linearized vector, Pacer fragment 1-412 PCR product, and mutant Pacer RH domain PCR product were mixed with Gibson Assembly master mix (NEB) in a 1:3:3 molar ratio prior to incubation at 50 °C for 15 minutes and transformation into XL10 E. coli. Plasmids were subsequently purified via overnight culture followed by purification via a commercial endotoxin-free spin midi prep kit (Qiagen).
To prepare for stable cell-line generation, mCherry-tagged full-length Rubicon, Pacer, and Pacer pRAB7A binding mutant were subcloned in a pCDH1 lentiviral vector. Briefly, both the pCHD1 vector and pmCherry-RH protein mammalian expression plasmids were digested using NheI and BamHI before being purified via gel electrophoresis. Bands corresponding to mCherry -RH protein were isolated and purified before being ligated into the pCDH1 backbone via T4 DNA polymerase. Subsequently, the final pCDH1 -RH protein construct was transformed into One Shot Stbl3 Chemically Competent E. Coli (Thermo Fisher) before being selected via antibiotic, and grown in an overnight culture. A maxiprep was performed to purify the plasmid (Qiagen), and the plasmid subsequently used for stable cell line generation via lentiviral transduction.
E. coli expressible constructs were verified via sequencing and SDS-PAGE of target proteins. Mammalian expressible constructs were verified via sequencing, visibility of fluorescent tag, and western blot against construct protein in a knockout background.

Protein Expression and Purification
Plasmids were transformed into BL21 star (DE3) E. coli (Agilent technologies), and individual colonies picked. For purification, 20 mL of overnight culture was inoculated, and then diluted into 4 L of lysogeny broth supplemented with maintenance antibiotics. When purifying MBP-Rubicon RH or MBP-Pacer RH, cultures were also supplemented with 150 µM ZnCl2. Cultures were grown at 37 C until OD600 = 0.55 was reached, and then cultures were chilled in ice water baths for 15 minutes before being induced with 350 µM isopropyl-β-D-thiogalactoside (IPTG). Induced cultures expressed protein at 18 C for 16-20 H before being harvested via centrifugation. Harvested cells were resuspended in a buffer consisting of 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, and 10 mM TCEP, and a tablet of cOmplete protease inhibitor cocktail (Roche). Cells were lysed via sonication, and the lysate was clarified by centrifugation at 34,500 g for 1 h at 4 °C.
MBP-tagged RH domain was isolated via passing cleared lysate over a gravity amylose resin column containing 5 mL of settled resin (NEB). Column was washed with 150 mL of buffer before eluting protein with wash buffer supplemented with 20 mM maltose. As a final polishing step, protein was concentrated down to 500 µL before being loaded onto a Superdex 75 Increase 10/300 GL size-exclusion column (Cytiva). Fractions were analyzed via SDS-PAGE, and pure fractions were pooled and concentrated before being concentrated and snap-frozen in liquid nitrogen GST-tagged RAB7A was similarly isolated via passing cleared lysate over a gravity column packed with 5 mL of Glutathione Sepharose 4B (GE Healthcare), followed by elution using 10 mM reduced glutathione dissolved in 50 mM Tris-HCl pH 8.0.. To cleave the GST tag, purified TEV protease was added in a 1:20 protease: RAB7A ratio by mass, and the solution was dialyzed overnight into a 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, 10 mM TCEP buffer. Protease and cleaved GST was removed from solution by passing the dialyzed and cleaved protein solution over a 10 mL Hispur Ni-NTA gravity column (Thermo Fisher) three times. Subsequently, the cleaved RAB7A was concentrated down to 500 µL, and further purified via size exclusion as with the purified RH domains.

Pulldowns
In a final volume of 30 µL, 20 µg of purified MBP-Rubicon RH domain was mixed with 20 µg of RAB7A in 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, 10 mM TCEP buffer. Samples were incubated on a rocker at room temperature for 1 H. 20 µL of 50% (v/v) amylose resin (NEB) was added to each sample, and samples were left to bind on rocker for an additional 30 min. Subsequently, supernatant was aspirated off, and beads were washed with 500 µL of icecold buffer for three total washes. Bound protein was eluted from beads via buffer supplemented with 20 mM maltose, and samples run on NuPAGE 4-12% Bis-Tris SDS-PAGE gel (Invitrogen), followed by staining with Coomassie G-250 brilliant blue.

Bead-binding
A 40 µL solution of 25% (v/v) amylose resin (NEB) was co-incubated with 500 nM MBP-RH domain and 4 µM Alexa Fluor 647-labeled RAB7A (life technologies, DOL = 0.9) in a buffer consisting of 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, 10 mM TCEP. Samples were incubated on rocker at room temperature for 1 H. Resin was collected using a tabletop centrifuge, supernatant was aspirated off, and resin was washed 3 times with 1 mL of room temperature buffer. Beads were resuspended in 200 µL buffer and transferred to an 8-well confocal imaging chamber (LabTek). Images were collected on a Ti2 Nikon confocal microscope with a 63 x Plan Apochromat 1.4 NA objective, and binding was quantified via the Alexa Fluor 647 channel. Bead fluorescence intensity was assessed in ImageJ via selecting an infocus bead, defining an ROI that includes the bead edge, and recording the maximum pixel intensity.
To assess degree of phosphorylation, a 15 µg pRAB7A sample was then buffer-exchanged into MQ water via dilution and concentration in a centrifugal concentrator, and samples were subsequently analyzed via electrophoresis on a SuperSep Phos-tag Precast gel (Wako Fujifilm). Complete band shift of the RAB7A band post phosphorylation confirms stoichiometric phosphorylation of the protein.

Isothermal titration calorimetry
To prepare proteins for isothermal titration calorimetry (ITC) binding studies, recombinantly purified RAB7A and MBP-Rubicon RH were co-dialyzed overnight at 4 C into a degassed buffer containing 50 mM HEPES 7.5, 150 mM NaCl, 2 mM MgCl2, and 10 mM TCEP. The next morning, RAB7A was concentrated to 800 µM, and MBP-Rubicon RH to 80 µM using a centrifugal concentrator. The concentrated protein solutions were again degassed via exposure to vacuum, allowed to come to room temperature, and loaded onto a Microcal PEAQ-ITC (Malvern Panalytical), with the MBP-Rubicon RH contained in the ITC chamber, and the RAB7A solution in the syringe. The titration was conducted, and the resulting binding curve fit using the PEAQ-ITC's native curve-fitting and analysis software.

Generation of knockout lines using CRISPR/Cas9 gene editing
RUBICON and PACER KO lines in this study were generated using CRISPR/Cas9 gene editing. CRISPR guide RNAs (gRNAs) targeting a common exon of splicing variants of each gene of interest were cloned into BBsI-linearised pSpCas9(BB)-2A-GFP vector (Addgene #48138) using NEBuilder® HiFi DNA Assembly (New England Biolabs) as described in the following protocol: https://doi.org/10.17504/protocols.io.j8nlkkzo6l5r/v1. gRNA constructs were transfected into HeLa S3 cells (https://www.atcc.org/products/ccl-2.2) with X-tremeGENE 9 (Roche) overnight. With fluorescence activated cell sorting (FACS), GFP-positive single cells were sorted into individual wells of 96-well plates. To identify knockout cell lines, single cell colonies were expanded and initially screened via western blot. In the absence of an antibody, the PACER KO line was confirmed via sequencing alone. The CRISPR targeted regions of the knockout clones and wild-type cells were amplified via PCR and sequenced using primers that annealed to the amplified product. Synthego ICE v2 CRISPR Analysis Tool (https://synthego.com/products/bioinformatics/crispr-analysis) was used to compare the sequencing data of the control and knockout cell lines to confirm the presence of indels.

Generation of stable cell lines
To prepare the retrovirus transduction solution, HEK293T cells were transfected for 6 H with pMRX-IP-based, pMRX-IB, or pMXs-based retroviral plasmids containing HaloTag autophagy reporters or HA-tagged Parkin, pCG-gag-pol, and pCG-VSV-G using TransIT-LT-1 transfection reagent (Mirus). Subsequently, the medium was replaced with DMEM, and the HEK cells were allowed to produce pseudotyped retrovirus for 3 days. The medium was harvested, cleared via centrifugation, and concentrated 10-fold via Lenti-X Concentrator solution (Takara). Retroviral solution was added to WT, Pacer knockout, and Rubicon knockout HeLa cells in a 12-well plate at 50% confluence. Cells were left to incubate for 24 H before swapping medium to fresh DMEM, and assessing transfection efficiency via fluorescence. Transfected cells were selected via incubation with puromycin or blasticidin depending on the transduction plasmid, and uniform express of autophagy reporters across cell types was confirmed via western blot and fluorescence microscopy

Immunofluorescence Imaging and Colocalization
Cells were seeded two days in advance on chambered glass slides treated with poly-L-lysine. Cells were allowed to grow to 80% confluence, and then transfected with mCherry-labeled RH proteins using lipofectamine 3000 (Thermo Fisher). Cells were allowed to recover for 48 H before being depolarized using CCCP for 1 H or starved via incubation in EBSS, then washed with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized via a 15 min incubation in Streptolysin O from Streptococcus pyogenes (Sigma Aldrich), washed three times with PBS, then blocked via a 30 min incubation in 2% BSA dissolved in PBS. Cells were washed and incubated for 1 H in a 1:200 primary solution of Anti-pRAB7A antibody (ab302494, Abcam).
To analyze and quantify colocalization, images were opened in FIJI, an open-source distribution of ImageJ. To subtract the background of each channel, a portion of the image expected to be dark, such as a nucleus, is defined and the average signal intensity measured. This average signal intensity is subtracted from the image as a whole using FIJI's Math>Subtract function. Subsequently, lasso ROIs are used to define individual cell boundaries, and the Mander's colocalization coefficient is calculated using the Coloc 2 plugin.

HaloTag processing assay
Cells stably expressing LC3-halo or Su9-halo were seeded for 80% confluence on the day of the assay in 12-well tissue culture plates. To pulse-label the reporter, cells were incubated for 20 min in 100 nM cell-permeable halotag conjugated to Janelia Fluor 549 dye (Promega) before washing twice with PBS. Cells were incubated in growth medium with 10 µM oligomycin and 5 µM antimycin for 6 H, in EBSS for 3 H, or in growth medium before harvesting cells via scraping. Cells were lysed, and the protein concentration was quantified in a plate reader using a Pierce BCA protein quantification kit (Thermo Fisher). For each condition, 20 µg of protein was loaded onto a gel for SDS-PAGE, and then the gels were imaged using a ChemiDoc fluorescent imager (Biorad).
Images of gels were imported into FIJI, and analyzed using the Gel Analyzer tool. Briefly, selections were defined that contained the bands of interest, and the integrated signal for each band was recorded. For mitophagy flux assays, the processed HaloTag band intensity was recorded, while for the starvation autophagy assays, the intensity of the processed HaloTag band as a fraction of the total Halo signal was calculated. For each treatment condition, the total signal was normalized to the signal produced by a rich and nondepolarizing medium control incubated for an equal amount of time. This value represents the fold enhancement in autophagy flux over baseline for each cell type and transfection.

Western blotting
Adherent cells to be analyzed were washed twice with PBS, and then scraped into a microcentrifuge tube. Cells were pelleted, lysed, and their concentration measured as performed for the HaloTag processing assay. 20 µg of protein was loaded into each lane. Protein was transferred to a nitrocellulose membrane (GE Healthcare) using a TransBlot Turbo transfer device (Biorad) and the device's default protocol. Post transfer, the membrane was cut lengthwise into strips to isolate bands of particular molecular weights before being washed with TBST and incubated at room temperature for 1 H in a 5% blocking buffer solution (Biorad). The western blot strips were then incubated overnight at 4 C in a solution consisting of the primary antibody diluted into blocking buffer. The next morning, the western blot strips were washed in TBST and incubated in a solution of secondary antibody diluted in blocking buffer for 1 H at room temperature. The strips were rigorously washed, and prepared for chemiluminescent imaging via incubation in SuperSignal West Femto ECL detection substrate (Thermo Fisher) for 5 minutes prior to imaging on a ChemiDoc (BioRad).

Autophagosome fluorescence microscopy live imaging
For live-cell imaging Pacer KO +HA-Parkin HeLa cells were first co-transfected with pCIG2-LAMP1-mNeon Green and pHaloTag-LC3B and either pmCherry, pmCherry-Pacer WT or pmCherry-Pacer K534N/R623T . Post 16-18 hr of transfection, cells were treated with Antimycin A and Oligomycin A for 4 hrs. For imaging, the culture media was replaced with Leibovitz's L-15 medium (Gibco 11415064) supplemented with 10% fetal bovine serum and 1% Glutamax along with Antimycin A and Oligomycin A. Imaging was performed on a PerkinElmer UltraView Vox spinning disk confocal on a Nikon Eclipse Ti Microscope with an Apochromat 100x 1.49 N.A. oilimmersion objective and a Hamamatsu CMOS ORCA Fusion (C11440-20UP) camera with VisiView (Visitron). mCherry was expressed to a higher level than either pmCherry-Pacer WT or pmCherry-Pacer K534N/R623T , resulting in higher fluorescent intensities per cell. Thus, the red channel for mCherry-expressing cells was acquired at a lower laser power and shorter exposure time than those used for both mCherry-Pacer WT and mCherry-Pacer K534N/R623T expressing cells; however, the LAMP1 and LC3B channels were imaged at the same laser power and exposure time across all three constructs. Imaging parameters were kept constant across biological replicates. Quantification was performed on individual slices from a Z-stack. Images from the LC3 and LAMP1 channels were thresholded and binarized using Yen thresholding in ImageJ. To quantify area of colocalization the 'AND' function from Image Calculator was used. Area of colocalization was then quantified using Analyze Particles.

Statistical analysis
All statistical analyses were performed as indicated in figure captions. Analysis performed in either Microsoft Excel or Graphpad Prism.   with mCherry labelled, full-length Rubicon, and then immunofluorescence was performed using general and pS72-specific RAB7A antibodies alongside an Alexa 488 secondary antibody. Rubicon-RAB7A colocalization was examined under rich conditions, and Rubicon-pRAB7A was quantified following 1h depolarization with 10 µM CCCP. (B) Line scans indicated in RAB7A (left) and pRAB7A (right) imaging trials. Each point on the line scan was baseline subtracted, and then normalized to the maximum signal on the line scan for each channel. (C) Colocalization was calculated as the above-threshold Mander's colocalization coefficient representing the fraction of Rubicon-positive pixels that are also RAB7A/pRAB7A positive. For each experiment, ~20 cells were analyzed in this way (transparent markers), and then the average of these 20 measurements constitutes one biological replicate (solid markers). The average of 3 biological replicates is represented by the horizontal bar marker. The p statistic was calculated using a one-tailed, paired T test comparing average colocalization fractions measured on the same day. (D) Confocal bead-binding assay, conducted as in Figure 2D. Amylose beads were incubated with either MBP-Rubicon RH or Mutant MBP-Rubicon RH N821K, T911R, K916R. (E) Quantification of panel D. Briefly, an ROI was defined on the edge of a bead to be analyzed, and the maximum pixel value in the ROI was recorded as the brightness of the bead. Each condition consists of 3 independent experiments in which the brightness of ~20 beads was measured and averaged. Error bars indicate standard deviation of these independent experiments.  Figure 2D, and quantified as in Figure 3D. Mutants of MBP-Pacer RH with the listed mutant were used in these experiments. (B) Quantification of panel A, conducted as in Figure 3E. Each experiment consists of ~20 different beads measured and averaged. This was repeated 3 times, and the mean of these biological replicates is plotted. Error bars indicate standard deviation of separate experiments. (C) Colocalization of mCherry-WT Pacer and mCherry-Mutant (K534N, R623T) Pacer. Cells were transfected with the listed Pacer construct, and depolarized for 1 H using CCCP. Immunofluorescence was performed using the pS72 specific antibody of RAB7A, and the Alexa 488 secondary antibody was used to mark pRAB7A. (D) Linescans indicated in panel C, calculated as in fig 3B. (E) Colocalization of WT and Mutant Pacer with pRAB7A, calculated as in Figure 3C.  Figure 1C. Parkin-expressing cells were treated with either complete medium, or complete medium supplemented with 10 µM Oligomycin A, 5 µM Antimycin A for 4 H. (B) Quantification of panel A, conducted as in Figure 1D. n = 3 for the uninduced condition, n = 9 for the induced conditions. Error bars indicate standard deviation, and p value was calculated using a one-tailed T test. (C) Pacer KO cells expressing Parkin and Su9-HaloTag reporter were stably transfected with either Pacer, the pRAB7A binding mutant Pacer characterized in Figure 4C-E, or empty vector, and then depolarized for 2 or 4 H. n = 3 independent experiments. P value was calculated using a one-factor ANOVA at 2 and 4 H. (D) The same cells used in panel C were exposed to the non-Parkin mitophagy inducer DFP for 8, 16, and 24 H, and analyzed as with previous Su9-Halo processing blots. (E) Quantification of panel D. n = 4 independent experiments. P value was calculated using a one factor ANOVA at each timepoint. (F) Pacer KO cells expressing LC3-Halo were stably transfected using either WT Pacer, pRAB7A-binding mutant Pacer, or empty vector, and the starvation autophagy flux assay was conducted as in fig 1a. (G) Quantification of panel F. Between 3 and 9 independent experiments were conducted for each timepoint. P value was calculated using a one-factor ANOVA at each timepoint.  Figure 4C-E. Cells were cotransfected with LAMP1-mNeon as a lysosomal marker and Halo-LC3 as a autophagosome marker. Cells were depolarized for 4 H in 10 µM Oligomycin A and 5 µM Antimycin A prior to live imaging and quantification in panels C, D, and E. (B) Pacer KO HeLa cells stably expressing Parkin and mCherry-WT Pacer/mCherry-Mutant Pacer were depolarized for 1 H in 10 µM CCCP. Cells were fixed, and imaged. Pacer puncta were counted using an automated particle counting program. 20 cells were analyzed and averaged in each biological replicate, and each cell's puncta count was normalized to the average untreated puncta count for that day. 3-4 of these experiments were performed and averaged. Colored dots indicate technical replicates, black dots indicate averages for each experimental day, black horizontal line indicates averages of biological replicates. P value calculated using a two-tailed T test of biological replicates. (C) Fold change in total autophagosome area. Cells imaged in panel A were masked and thresholded, and the total LC3 positive area was quantified. The total area for each cell was normalized to the average area for the mCherry transfected condition, and these values were averaged to calculate the value for a single biological replicate. This process was repeated in 4 independent experiments. P values were determined using Tukey's multiple comparison test. (D) Fold change in LC3+/LAMP1+ positive area, determined as in panel C. (E) Fraction of total LC3 area that is also positive for LAMP1 as a metric for autophagosome-lysosome fusion efficiency, analysis performed as in panel C.