The cyclophilin homolog ninaA is required in the secretory pathway

In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.     

Golgi-bound Rab34 is a novel member of the secretory pathway

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.     

The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

The genes for two new P-type ATPases, PMR1 and PMR2, have been identified in yeast. A comparison of the deduced sequences of the PMR proteins with other known ion pumps showed that both proteins are very similar to Ca2+ ATPases. PMR1 is identical to SSC1, a gene previously identified by its effect on secretion of some foreign proteins from yeast. Proteins secreted from pmr1 mutants lack the outer chain glycosylation that normally results from passage through the Golgi. Loss of PMR1 function suppresses the lethality of ypt1-1, a mutation that blocks the secretion pathway. These data suggest that PMR1 functions as a Ca2+ pump affecting transit through the secretory pathway.     

Protein processing within the secretory pathway.

Endoproteolytic cleavage of hormone and neuropeptide precursors, as well as many complex proteins, such as coagulation factors and viral glycoproteins, is a key process in the generation of bioactive polypeptides. These cleavages typically occur at the dibasic amino acid residues Lys-Arg or Arg-Arg. The enzymes responsible for the processing belong to a newly discovered family of serine proteases related to the bacterial subtilisins. These include PACE (furin), PC1/PC3, PC2 and PACE4, which have all been characterized functionally and structurally.     

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.     

Bacterial interactions with the eukaryotic secretory pathway

Pathogenic bacteria exploit a wide variety of host cellular processes to adhere to, invade, replicate within and damage host cells. One such process is the eukaryotic secretory pathway, in which proteins and lipids are modified and transported from the endoplasmic reticulum through the Golgi network to the plasma membrane and other cellular destinations. Certain bacteria secrete toxins that utilise this transport pathway to reach their cellular targets. Some intracellular pathogens, including Legionella, Brucella and Chlamydia, engage other steps of the pathway to establish intracellular replicative organelles. Recent work has implicated specific virulence proteins of enterohaemorrhagic Escherichia coli and Salmonella enterica in secretory pathway interactions.     

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.     

Myristoylation of proteins in the yeast secretory pathway.

Protein myristoylation was investigated in the yeast secretory pathway. Conditional secretory mutations were used to accumulate inteRmediaries in the pathway between the endoplasmic reticulum and Golgi (sec 18, 20), within the Golgi (sec 7), and between the Golgi and plasma membrane (sec 1, 3, 4, 5, 6, 8, 9). The accumulation of vesicles was paralleled by the enrichment of a defined subset of proteins modified either via ester or amide linkages to myristic acid: Myristoylated proteins of 21, 32, 49, 56, 75, and 136 kDa were enriched between the endoplasmic reticulum and Golgi; proteins of 21, 32, 45, 56, 75, 136 kDa were enriched by blocks within the Golgi; and proteins of 18, 21, 32, 36, 49, 68, and 136 kDa were trapped in a myristoylated form by blocks between the Golgi and plasma membrane. This enrichment of myristoylated proteins was reversed upon returning the cells to the permissive temperature for secretion. The fatty acid was linked to the 21-kDa protein via a hydroxylamine-resistant amide linkage (N-myristoylation) and to the proteins of 24, 32, 49, 56, 68, 136 kDa via hydroxylamine-labile ester linkage (E-myristoylation). In addition, myristoylated proteins of 21, 56, and 136 kDa were glycosylated via amino linkages to asparagine. This suggests they are exposed to the lumen of the secretory pathway. Three proteins (24, 32, and 56) were E-myristoylated in the presence of protein synthesis inhibitors, indicating this modification can occur posttranslationally. After using cycloheximide to clear protein passengers from the secretory pathway the 21-, 32-, and 56-kDa proteins continued to accumulate in a myristoylated form when vesicular transport was blocked between the Golgi and plasma membrane. These data suggest that myristoylation occurs on a component of the secretory machinery rather than on a passenger protein.     

Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins

We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.     

The secretory pathway Ca-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca²⁺/Mn²⁺ pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.