David Sheff

Cytoplasmic coat proteins involved in endosome function

Endosomes are intermediates for a complex series of sorting and transport events that occur during receptor-mediated endocytosis. These involve the recognition of targeting determinants on the cytoplasmic domains of many membrane proteins as well as the formations of specific transport vesicles. Accordingly, endosome function is likely to be governed by the regulated assembly of cytoplasmic coat complexes. We have found that, in vitro, endosomes recruit a characteristic set of cytoplasmic proteins in a GTP gamma S-stimulated and brefeldin A-sensitive fashion. Among these are members of the COP-I and ARF families of coat proteins. In addition, endosomes were also found to assemble distinct, clathrin-like coats. Since microinjection of antibodies to beta-COP inhibits the entry of enveloped viruses via the endocytic pathway, it is apparent that the recruitment of COP-I or COP-I-related proteins plays an important role in the function of endosomes in intact cells.

Golgi biogenesis in Toxoplasma gondii

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Transferrin receptor recycling in the absence of perinuclear recycling endosomes

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the mu1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology

Depletion of EHD3 affects sorting in endosomes by altering the kinetics and route of receptor recycling to the plasma membrane. Here we demonstrate that siRNA knockdown of EHD3, or its interaction partner rabenosyn-5, causes redistribution of sorting nexin 1 (SNX1) to enlarged early endosomes and disrupts transport of internalized Shiga toxin B subunit (STxB) to the Golgi. Moreover, under these conditions, Golgi morphology appears as a series of highly dispersed and fragmented stacks that maintain characteristics of cis-, medial- and trans-Golgi membranes. Although Arf1 still assembled onto these dispersed Golgi membranes, the level of AP-1 γ-adaptin recruited to the Golgi was diminished. Whereas VSV-G-secretion from the dispersed Golgi remained largely unaffected, the distribution of mannose 6-phosphate receptor (M6PR) was altered: it remained in peripheral endosomes and did not return to the Golgi. Cathepsin D, a hydrolase that is normally transported to lysosomes via an M6PR-dependent pathway, remained trapped at the Golgi. Our findings support a role for EHD3 in regulating endosome-to-Golgi transport, and as a consequence, lysosomal biosynthetic, but not secretory, transport pathways are also affected. These data also suggest that impaired endosome-to-Golgi transport and the resulting lack of recruitment of AP-1 γ-adaptin to Golgi membranes affect Golgi morphology.

Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y33RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAMs ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr33. This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.

Cloning, expression, and localization of a novel γ-adaptin-like molecule

We describe the cloning, expression, and localization of γ2‐adaptin, a novel isoform of γ‐adaptin. The predicted human and mouse γ2‐adaptin proteins are ∼90 kDa and 64.4% and 61.7% identical to γ‐adaptin, respectively. γ2‐Adaptin was localized to the Golgi, its localization distinct from γ‐adaptin. The membrane association of γ‐ and γ2‐adaptin could further be distinguished by differential sensitivity to the fungal metabolite brefeldin A, γ2‐adaptin binding being insensitive to drug treatment. Together, these results suggest that γ2‐adaptin plays a role in membrane transport distinct from that played by γ‐adaptin.

Regulator of G Protein Signaling 6 (RGS6) Protein Ensures Coordination of Motor Movement by Modulating GABAB Receptor Signaling

Background: GABABR signaling blocks neuronal firing ensuring appropriate cerebellar cortex output. Results: Loss of RGS6 results in ataxia rescued by a GABABR antagonist and enhanced GABABR-GIRK current in neurons. Conclusion: RGS6 is an essential component of GABA signaling in cerebellum and required for motor coordination. Significance: RGS6 dysregulation could result in cerebellar ataxia, and thus, it might represent a novel target for pharmacological intervention. γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABAB receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABABR signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ5 and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABABR antagonist. RGS6−/− mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABABR, and GIRK channel subunits, and cerebellar granule neurons from RGS6−/− mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABABR signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.

RalA and RalB differentially regulate development of epithelial tight junctions

The closely related GTPases RalA and RalB are required for assembly of tight junction gate, but not fence, function. These activities depend on direct binding to the exocyst complex. Whereas RalA–exocyst complexes are required for exocytosis of junction proteins, RalB–exocyst complexes are required for endocytosis of these components.