The transcription factor hiF1 is mostly regulated by the oxygen-dependent proteasomal degradation of the labile subunit hiF1A. recent data showed degradation of hiF1A in the lysosome through chaperone-mediated autophagy (cMA). however the molecular mechanism involved has not been elucidated. This study shows that the KFerQ-like motif, that has been identified in all cMA substrates, is required to mediate the interaction between hiF1A and the chaperone hSpA8. Moreover, mutations in the KFerQ-like motif of hiF1A preclude the interaction with the cMA receptor LAMp2A, thus inhibiting its lysosomal degradation. importantly, we show for the first time that the ubiquitin ligase STUB1 is required for degradation of hiF1A in the lysosome by cMA. indeed, mutations in STUB1 that inhibit either the ubiquitin ligase activity or its ability to bind to hSpA8, both prevent degradation of hiF1A by cMA. Moreover, we show that hiF1A binds to and is translocated into intact lysosomes isolated from rat livers. This new pathway for degradation of hiF1A does not depend on the presence of oxygen and is activated in response to nutrient deprivation such that the levels of hiF1A bound to cMA positive lysosomes significantly increase in starved animal livers and the binding of hiF1A to LAMp2A increases in response to serum deprivation. Moreover, excessive degradation of hiF1A by cMA compromises cells' ability to respond to and survive under hypoxia, suggesting that this pathway might be of pathophysiological importance in conditions that combine hypoxia with starvation. leucinylleucinylleucinal/proteasome inhibitor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; RCC, renal cell carcinoma; Serpinb, serpin peptidase inhibitor, clade B/Ovalbumin; SLC2A1, solute carrier family 2 (facilitated glucose transporter)/glucose transporter; STUB1, STIP1 homology and U-box containing protein 1; TPR, tetratricopeptide repeat; UPS, ubiquitin-proteasome system; VEGFA, vascular endothelial growth factor A; u-box, modified RING finger domain lacking the metal-chelating residues

Griscelli syndrome (GS) patients and the corresponding mouse model ashen exhibit defects mainly in two types of lysosome-related organelles, melanosomes in melanocytes and lytic granules in CTLs. This disease is caused by loss-of-function mutations in RAB27A, which encodes 1 of the 60 known Rab GTPases, critical regulators of vesicular transport. Here we present evidence that Rab27a function can be compensated by a closely related protein, Rab27b. Rab27b is expressed in platelets and other tissues but not in melanocytes or CTLs. Morphological and functional tests in platelets derived from ashen mice are all within normal limits. Both Rab27a and Rab27b are found associated with the limiting membrane of platelet-dense granules and to a lesser degree with α-granules. Ubiquitous transgenic expression of Rab27a or Rab27b rescues ashen coat color, and melanocytes derived from transgenic mice exhibit widespread peripheral distribution of melanosomes instead of the perinuclear clumping observed in ashen melanocytes. Finally, transient expression in ashen melanocytes of Rab27a or Rab27b, but not other Rab's, restores peripheral distribution of melanosomes. Our data suggest that Rab27b is functionally redundant with Rab27a and that the pathogenesis of GS is determined by the relative expression of Rab27a and Rab27b in specialized cell types.

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle.Fundação para a Ciência e Tecnologia grants: (PTDC/IMI-MIC/1142/2012, SFRH/BPD/94204/2013, SFRH/BPD/62982/2009, IF/00899/2013); Fundação Calouste Gulbenkian; Instituto Gulbenkian de Ciência

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

On the path to successful immunotherapy of hematopoietic tumors, ␥␦ T cells offer great promise because of their human leukocyte antigen (HLA)-unrestricted targeting of a wide variety of leukemias/ lymphomas. However, the molecular mechanisms underlying lymphoma recognition by ␥␦ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to ␥␦ T cell-mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all V␥9 ؉ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by ␥␦ T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to ␥␦ T cell-based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials. (Blood. 2010;115:2407-2411)

Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.

Encarnação et al. show that Rab3a, together with its newly identified effector NMHC IIA, mediates the positioning of peripheral lysosomes in nonsecretory cells, thereby promoting lysosome exocytosis and plasma membrane repair.