Rab27a and MyRIP regulate the amount and multimeric state of VWF released from endothelial cells.

Endothelial cells contain cigar-shaped secretory organelles called Weibel-Palade bodies (WPBs) that play a crucial role in both hemostasis and the initiation of inflammation. The major cargo protein of WPBs is von Willebrand factor (VWF). In unstimulated cells, this protein is stored in a highly multimerized state coiled into protein tubules, but after secretagogue stimulation and exocytosis it unfurls, under shear force, as long platelet-binding strings. Small GTPases of the Rab family play a key role in organelle function. Using siRNA depletion in primary endothelial cells, we have identified a role for the WPB-associated Rab27a and its effector MyRIP. Both these proteins are present on only mature WPBs, and this rab/effector complex appears to anchor these WPBs to peripheral actin. Depletion of either the Rab or its effector results in a loss of peripheral WPB localization, and this destabilization is coupled with an increase in both basal and stimulated secretion. The VWF released from Rab27a-depleted cells is less multimerized, and the VWF strings seen under flow are shorter. Our results indicate that this Rab/effector complex controls peripheral distribution and prevents release of incompletely processed WPB content.

Endosomal and non-endosomal functions of ESCRT proteins.

The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.

Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors.

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.

Cloning and subcellular localization of a human phosphatidylinositol 3-phosphate 5-kinase, PIKfyve/Fab1.

Yeast Fab1 is a phosphatidylinositol 3-phosphate 5-kinase involved in endocytic membrane traffic and vacuole homeostasis. Here we have cloned and sequenced the cDNA for the human homologue of Fab1, PIKfyve. The cDNA has an open reading frame of 6294 bp and encodes a 2098-amino acid protein with a calculated molecular mass of 237 kDa, containing a phosphatidylinositol 3-phosphate-binding FYVE domain, a DEP domain, a chaperonin-like domain, and a phosphoinositide kinase domain. The human genome contains a single PIKfyve gene, which comprises 38 exons on chromosomal locus 2q34. PIKfyve is expressed as a single molecular species in a number of human cell lines derived from different tissues. The exogenously expressed protein was found to localize mainly to early endosomes containing two other FYVE domain proteins, EEA1 and Hrs. The endosomal membrane localization of PIKfyve was studied in more detail by examining cells transfected with a constitutively active mutant of the small GTPase Rab5, whose expression results in the enlargement of early endosomes. We show that PIKfyve is distributed in microdomains that are distinct from those occupied by EEA1 and Hrs.

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases.

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.

Calpain cleavage of the B isoform of Ins(1,4,5)P3 3-kinase separates the catalytic domain from the membrane anchoring domain.

Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport.

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.