Rab2 stimulates LC3 lipidation on secretory membranes by noncanonical autophagy.

The Golgi complex is a highly dynamic organelle that regulates various cellular activities and yet maintains a distinct structure. Multiple proteins participate in Golgi structure/organization including the small GTPase Rab2. Rab2 is found on the cis/medial Golgi compartments and the endoplasmic reticulum-Golgi intermediate compartment. Interestingly, Rab2 gene amplification occurs in a wide range of human cancers and Golgi morphological alterations are associated with cellular transformation. To learn how Rab2 'gain of function' influences the structure/activity of membrane compartments in the early secretory pathway that may contribute to oncogenesis, NRK cells were transfected with Rab2B cDNA. We found that Rab2B overexpression had a dramatic effect on the morphology of pre- and early Golgi compartments that resulted in a decreased transport rate of VSV-G in the early secretory pathway. We monitored the cells for the autophagic marker protein LC3 based on the findings that depressed membrane trafficking affects homeostasis. Morphological and biochemical studies confirmed that Rab2 ectopic expression stimulated LC3-lipidation on Rab2-containing membranes that was dependent on GAPDH and utilized a non-canonical LC3-conjugation mechanism that is nondegradative. Golgi structural alterations are associated with changes in Golgi-associated signalling pathways. Indeed, Rab2 overexpressing cells had elevated Src activity. We propose that increased Rab2 expression facilitates cis Golgi structural changes that are maintained and tolerated by the cell due to LC3 tagging, and subsequent membrane remodeling triggers Golgi associated signaling pathways that may contribute to oncogenesis.

GAPDH binds Akt to facilitate cargo transport in the early secretory pathway.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes numerous post-translational modifications, which impart new function and influence intracellular location. For example, atypical PKC ι/λ phosphorylates GAPDH that locates to vesicular tubular clusters and is required for retrograde membrane trafficking in the early secretory pathway. GAPDH is also required in the endocytic pathway; substitution of Pro234 to Ser (Pro234Ser) rendered CHO cells defective in endocytosis. To determine if GAPDH (Pro234Ser) could inhibit endoplasmic reticulum to Golgi trafficking, we introduced the recombinant mutant enzyme into several biochemical and morphological transport assays. The mutant protein efficiently blocked vesicular stomatitis virus-G protein transport. Because GAPDH binds to microtubules (MTs), we evaluated MT binding and MT intracellular distribution in the presence of the mutant. Although these properties were not changed relative to wild-type, GAPDH (Pro234Ser) altered Golgi complex morphology. We determined that the GAPDH point mutation disrupted association between the enzyme and the serine/threonine kinase Akt. Interestingly Rab1, which functions in anterograde-directed trafficking, stimulates GAPDH-Akt association with membranes in a quantitative binding assay. In contrast, Rab2 does not stimulate GAPDH-Akt membrane binding but instead recruits GAPDH-aPKC. We propose a mechanism whereby the association of GAPDH with Akt or with aPKC serves as a switch to discriminate between anterograde directed cargo and recycling cargo retrieved back to the ER, respectively.

The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation.

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders.

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.

The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.

Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation.

Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration.

Rab2 utilizes glyceraldehyde-3-phosphate dehydrogenase and protein kinase C{iota} to associate with microtubules and to recruit dynein.

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Ciota (aPKCiota) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCiota in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCiota were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCiota. Because GAPDH binds to the carboxyl terminus of alpha-tubulin, we characterized the distribution of tyrosinated/detyrosinated alpha-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated alpha-tubulin; however, aPKCiota was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCiota-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.

A GAPDH mutant defective in Src-dependent tyrosine phosphorylation impedes Rab2-mediated events.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2-mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2-dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane-binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose-dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus-G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C iota/lambda to GAPDH Y41F, which reduces beta-coat protein association with the VTC and subsequent formation of Rab2-mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.

Src-dependent aprotein kinase C iota/lambda (aPKCiota/lambda) tyrosine phosphorylation is required for aPKCiota/lambda association with Rab2 and glyceraldehyde-3-phosphate dehydrogenase on pre-golgi intermediates.

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and aPKCiota/lambda and GAPDH interact. Based on the reports demonstrating aPKCiota-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCiota/lambda and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCiota/lambda on the VTC. To determine the consequence of aPKCiota/lambda tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCiota/lambda. Since Rab2 requires aPKCiota/lambda for the downstream recruitment of beta-coat protein (beta-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of beta-COP. Like aPKCiota/lambda, the membranes contained a negligible amount of beta-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCiota/lambda facilitates aPKCiota/lambda association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of beta-COP and release of Rab2-mediated retrograde-directed vesicles.

Rab2 purification and interaction with protein kinase C iota/lambda and glyceraldehyde-3-phosphate dehydrogenase.

The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway. Rab2 associates with vesicular tubular clusters (VTCs) located between the endoplasmic reticulum (ER) and the Golgi complex. VTCs function as transport intermediates and sort anterograde-directed cargo from recycling proteins. Rab2 selectively recruits atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate (GAPDH) to VTCs where aPKCiota/lambda phosphorylates GAPDH. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and this interaction ultimately results in COPI recruitment and the release of retrograde-directed vesicles. This chapter describes a protocol to purify recombinant Rab2 from Rab2 cDNA transformed bacteria and methods to assess recombinant Rab2 biological activity. Additionally, in vivo and in vitro assays are outlined that are employed to demonstrate Rab2 interaction with the downstream effectors aPKCiota/lambda and GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity.

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.

Rab2 interacts directly with atypical protein kinase C (aPKC) iota/lambda and inhibits aPKCiota/lambda-dependent glyceraldehyde-3-phosphate dehydrogenase phosphorylation.

Atypical protein kinase C iota/lambda (PKCiota/lambda) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCiota/lambda phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCiota/lambda and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCiota/lambda membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCiota/lambda and that this interaction occurs through the Rab2 amino terminus (residues 1-19) and the PKCiota/lambda regulatory domain. A mutant lacking the PKCiota/lambda binding domain (Rab2N'Delta19) was functionally characterized. In contrast to Rab2, Rab2N'Delta19 failed to recruit PKCiota/lambda to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCiota/lambda to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N'Delta19. Rab2 inhibited PKCiota/lambda-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCiota/lambda-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCiota/lambda interaction.

The N'-terminal domain of glyceraldehyde-3-phosphate dehydrogenase of the apicomplexan Plasmodium falciparum mediates GTPase Rab2-dependent recruitment to membranes.

Spatial and temporal distribution of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (pfGAPDH) and aldolase (pfAldolase) of Plasmodium falciparum were investigated using specific mAbs and indirect immunofluorescence analysis (IFA). Both glycolytic enzymes were co-localized during ring and trophozoite stages of both liver and asexual blood stage parasites. During schizogony, pfGAPDH became associated with the periphery of the parasites and eventually accumulated in the apical region of merozoites, while pfAldolase showed no segregation. Subcellular fractionation experiments demonstrated that pfGAPDH was found in both the membrane-containing pellet and the supernatant fraction of parasite lysates. In contrast, pfAldolase was only found in the supernatant fraction. A quantitative binding assay showed that pfGAPDH could be recruited to HeLa cell microsomal membranes in response to mammalian GTPase Rab2, indicating that Rab2-dependent recruitment of cytosolic components to membranes is conserved in evolution. Two overlapping fragments of pfGAPDH (residues 1-192 and 133-337) were evaluated in the microsomal binding assay. We found that the N'-terminal fragment competitively inhibited Rab2-stimulated pfGAPDH recruitment. Thus, the domain mediating the evolutionarily conserved Rab2-dependent membrane recruitment is located in the N'-terminus of GAPDH. Together, these results suggest that pfGAPDH exerts non-glycolytic function(s) in P. falciparum, possibly including a role in vesicular transport and biogenesis of apical organelles.

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates.

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway.

The small GTPase Rab2 immunolocalizes to vesicular tubular clusters (VTCs) that function as transport complexes carrying cargo between the endoplasmic reticulum and the Golgi complex. Our previous studies showed that Rab2 promotes vesicle formation from VTCs and that the released vesicles are enriched in beta-coat protein, protein kinase C iota/lambda (PKCiota/lambda), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCiota/lambda kinase activity was necessary for vesicle formation, a search was initiated to identify the substrate(s) that potentiate Rab2 function within VTCs. In this study, we found that PKCiota/lambda phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCiota/lambda regulatory domain. Based on numerous observations that show (beta-COP) GAPDH associates with cytoskeletal elements, we examined the role of phospho-GAPDH in promoting microtubule (MT) binding to membrane. Using a quantitative microsomal binding assay, we found that membrane association of beta-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCiota/lambda kinase activity. Furthermore, normal rat kidney cells transfected with a constitutively activated form of Rab2 (Q65L) or with our anti-GAPDH polyclonal antibody displayed a dramatic change in MT organization. These combined results suggest that Rab2 stimulated PKCiota/lambda and GAPDH recruitment to VTCs, and the subsequent PKCiota/lambda phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.