Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.

Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.

Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells.

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.

Analysis of low abundance membrane-associated proteins from rat pancreatic zymogen granules.

Zymogen granules (ZG) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging, and regulated apical secretion of digestive enzymes. As there is a critical need for further understanding of the key processes in regulated secretion to develop new therapeutic options in medicine, we applied a suborganellar proteomics approach to identify peripheral membrane-associated ZG proteins. We focused on the analysis of a "basic" group (pH range 6.2-11) with about 46 spots among which 44 were identified by tandem mass spectrometry. These spots corresponded to 16 unique proteins, including rat mast cell chymase (RMCP-1) and peptidyl-prolyl cis-trans isomerase B (PpiB; cyclophilin B), an ER-resident protein. To confirm that these proteins were specific to zymogen granules and not contaminants of the preparation, we conducted a series of validation experiments. Immunoblotting of ZG subfractions revealed that chymase and PpiB behaved like bona fide peripheral membrane proteins. Their expression in rat pancreas was regulated by feeding behavior. Ultrastructural and immunofluorescence studies confirmed their ZG localization. Furthermore, a chymase-YFP fusion protein was properly targeted to ZG in pancreatic AR42J cells. Interestingly, for both proteins, proteoglycan-binding properties have been reported. The importance of our findings for sorting and packaging during ZG formation is discussed.

The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.

The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.

Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism.

BACKGROUND: Human chorionic gonadotropin/luteinizing hormone (hCG/LH) function in the male is mediated by the LH receptor (LHR) and is crucial for the normal development of internal and external genitalia. We report a 46, XY patient who presented at the age of 16 with a female phenotype and delayed puberty. Gonads were located bilaterally in the inguinal canal, removed surgically and showed hypoplastic Leydig cells. Immunostaining for the LHR revealed that some Leydig cell progenitors were positive, while others were negative, reflecting different developmental stages of Leydig cell maturation. METHODS AND RESULTS: Molecular analysis of the LHR was performed on DNA extracted from blood samples of the patient, her parents and sister. The 11 exons of the LHR gene were amplified by PCR and subjected to further single stranded conformation polymorphism (SSCP) analysis. Aberrant migration patterns were observed in exon 7. Upon sequencing, a homozygous T to G transversion was identified, resulting in a F194V substitution located in the extracellular domain. The parents and sister were heterozygous carriers of this mutation. Functional studies in transiently transfected COS-7 cells with the F194V LHR mutation showed the lack of cAMP production upon hCG stimulation, indicating complete inactivation of the receptor due to impaired trafficking of the receptor to the membrane. The mutation is located within a stretch of five amino acids Ala (A)-Phe (F)-Asn (N)-Gly (G)-Thr (T), highly conserved in glycoprotein hormone receptors. For the follicle-stimulating hormone (FSH) receptor (FSHR) loss-of-function mutations have been allocated to this region, a homozygous A189V mutation resulting in a resistant ovary syndrome and impaired spermatogenesis and a heterozygous N191I mutation with no apparent phenotype. Further mutational and functional analysis of the AFN region in the LHR and FSHR revealed that the integrity of this amino acid sequence is crucial for receptor function.